Acta Biomaterialia 7 (2011) 751–758

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Acta Biomaterialia

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Resveratrol-conjugated poly-e-caprolactone facilitates in vitro mineralizationand in vivo bone regeneration

Yan Li a,⇑, Staffan Dånmark b, Ulrica Edlund b, Anna Finne-Wistrand b, Xu He a, Maria Norgård d, Eva Blomén c,Kjell Hultenby c, Göran Andersson d, Urban Lindgren a

a Department for Clinical Science, Intervention and Technology (CLINTEC), Division of Orthopedics, Karolinska Institute, Stockholm, Swedenb Fibre and Polymer Technology, School of Chemical Science and Engineering, Royal Institute of Technology, Stockholm, Swedenc Department of Laboratory Medicine, Division of Clinical Research Centre, Karolinska University Hospital Huddinge, Karolinska Institute, Stockholm, Swedend Department of Laboratory Medicine, Division of Pathology, Karolinska University Hospital Huddinge, Karolinska Institute, Stockholm, Sweden

a r t i c l e i n f o

Article history:Received 4 April 2010Received in revised form 30 August 2010Accepted 8 September 2010Available online 16 September 2010

Keywords:ResveratrolBiodegradable scaffoldsBone regenerationVapor phase graftingTissue engineering

1742-7061/$ - see front matter � 2010 Acta Materialdoi:10.1016/j.actbio.2010.09.008

⇑ Corresponding author. Tel.: +46 8 58583869; fax:E-mail address: yan.li@ki.se (Y. Li).

a b s t r a c t

Incorporation of osteoinductive factors in a suitable scaffold is considered a promising strategy for gen-erating osteogenic biomaterials. Resveratrol is a polyphenol found in parts of certain plants, includingnuts, berries and grapes. It is known to increase DNA synthesis and alkaline phosphatase (ALP) activityin osteoblasts and to prevent femoral bone loss in ovariectomized (OVX) rats. In the present study resve-ratrol was coupled through a hydrolysable covalent bond with the carboxylic acid groups in porous poly-e-caprolactone (PCL) surface grafted with acrylic acid (AA). The osteogenic effect of this new scaffold wasevaluated in mesenchymal cell culture and in the rat calvarial defect model. We found that the incorpo-ration of resveratrol caused increased ALP activity of rat bone marrow stromal cells and enhanced min-eralization of the cell–scaffold composites in vitro. After 8 weeks the calvarial defects implanted withresveratrol-conjugated PCL displayed a higher X-ray density than the defects implanted with controlPCL. Bone-like structures, positively immunostained for bone sialoprotein, were shown to be more exten-sively formed in the resveratrol-conjugated PCL. These results show that incorporation of resveratrol intothe AA-functionalized porous PCL scaffold led to a significant increase in osteogenesis.

� 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

A plethora of local growth factors have been identified withosteotrophic properties, such as bone morphogenetic proteins(BMPs), fibroblast growth factors (FGFs) and insulin-like growth fac-tors I and II (IGF I/II) [1]. In bone tissue engineering these factors havebeen combined with different materials, ranging from inorganicbone graft substitutes (e.g. hydroxyapatite, calcium phosphate-based cements) and natural tissue components (e.g. collagen, hyalu-ronan) to different types of synthetic polymers, with the purpose ofcreating osteoinductive or osteoconductive scaffolds [2]. Althoughpromising results have been noted, these factors are relativelyunstable during industrial processing, making their applicationcostly and the techniques demanding [3]. Therefore, the search forother osteotrophic substances has become an intriguing route tothe development of bone regenerative scaffolds.

Resveratrol is a polyphenol found in parts of certain plants,including nuts, berries and grapes [4,5]. It is known to exert avariety of health benefits in mammals, among which the cancer

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+46 8 58582224.

chemopreventive and cardio- and neuro-protective propertieshave attracted most attention [6]. Its osteotrophic effects were firstreported by Mizutani et al., who showed that resveratrol dose-dependently increased DNA synthesis and alkaline phosphatase(ALP) activity in osteoblasts [7]. Since these effects could beblocked by tamoxifen, an anti-estrogen, they concluded that resve-ratrol acted via estrogen receptors (ER) [7]. This notion was sub-stantiated by their subsequent in vivo study, which showed thatresveratrol prevented femoral bone loss in ovariectomized (OVX)rats [8]. A recent study by Su et al. [9] further clarified the under-lying mechanism of resveratrol-mediated bone protection. Theyfound that resveratrol stimulated BMP-2 production by osteoblaststhrough Src kinase-dependent ER activation and increased the ser-um concentration of BMP-2 in OVX rats [9]. Besides the ER/BMP-2pathway, our previous findings indicated that resveratrol epigenet-ically modified the gene expression in mesenchymal stem cells(MSCs) through activation of Sirt1, a NAD-dependent histonedeacetylase, which resulted in increased osteoblast differentiationin MSC cultures [10].

Despite the extensive biological effects in lower organisms,resveratrol is known to be quickly metabolized in the human body,resulting in an extremely short plasma half-life and very low

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752 Y. Li et al. / Acta Biomaterialia 7 (2011) 751–758

bioavailability [11]. Recently, attempts at developing synthetic car-rier systems for target-specific delivery of resveratrol have been re-ported [12–15], however, their effects have not been evaluatedusing in vivo models. Synthetic biodegradable polymers are oftenused as protein carriers since their structure, geometry, porosity,mechanical properties, surface properties and degradation kineticscan be tailored according to requirements [16–20]. We have previ-ously invented a vapor phase grafting method for surface function-alization of synthetic polymers in a non-degrading, solvent-freefashion [21]. This method for covalent immobilization of a hydro-philic graft chain on the substrate surface has been shown to notonly significantly increase hydrophilicity and biocompatibility ofthe material [21–23] but also facilitate the attachment of bioactivemolecules [24]. In the present study we functionalized the innerand outer surfaces of a porous poly-e-caprolactone (PCL) scaffoldby vapor phase grafting with acrylic acid (AA). The graft chain car-boxylic acid pendant groups were then covalently bonded withresveratrol through a hydrolysable linkage. The biocompatibilityand osteoconductive effect of this new scaffold were evaluated inrat bone marrow stromal cell cultures and rat calvarial defectmodels.

2. Materials and methods

2.1. Reagents

Modified Eagle’s medium alpha (a-MEM), fetal bovine serum(FBS), L-glutamine and gentamycin were purchased from Invitro-gen (Life Technologies, Paisley, UK). Resveratrol was purchasedfrom Sigma–Aldrich. Polymer scaffolds were produced from poly-e-caprolactone (PCL) (Mn = 80000, Aldrich) using chloroform (Lab-scan) and NaCl (Fischer, ground and dried before use). The polymerstructure and molecular weight was verified by 1H NMR and sizeexclusion chromatography (SEC) analyses. Benzophenone (BPO)(>99% pure, Acros), acrylic acid (AA) (Aldrich, distilled at reducedpressure before use), resveratrol (99% GC, Sigma), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Aldrich)and ethanol (EtOH) (95%, Kemetyl AB, Sweden) were used for sur-face functionalization the following chemicals. A 0.1 M MES buffer,pH 6.0, was prepared from 2-[N-morpholino]ethanesulfonic acidhemisodium salt (MES) (Sigma–Aldrich, Germany).

2.2. Preparation of porous PCL scaffolds

Porous PCL scaffolds were prepared as previously described [25]in a salt leaching process using NaCl as the porogen. The porogenwas ground once in a mortar prior to use. No size discriminationwas carried out. After extensive drying circular pieces of 5 mmdiameter were cut for in vivo rat calvarial defect implantationand samples with a diameter of 16 mm were cut for cell culture.The pieces had a thickness of 2.5 mm. They were stored under vac-uum until use. The material had a broad pore size range and anopen porosity of 77%, as determined by the Swedish Ceramic Insti-tute. The polymer structure was verified by 1H NMR, recorded at500 MHz in a Bruker DMX 500 spectrometer using Bruker soft-ware. The samples were dissolved in CHCl3 in 5 mm diameter sam-ple tubes. Non-deuterated CHCl3 (d = 7.26 ppm) was used as aninternal standard.

2.3. Covalent grafting of porous scaffolds

Our previously developed vapor phase grafting method wasused for covalent grafting [21–23] of AA on the inner and outersurfaces of the porous scaffolds. Grafting was performed at a molarratio of monomer to initiator of 10:1 at 30 ± 0.2 �C for 15 min. The

grafted scaffolds were rinsed extensively in deionized water for 3–4 h and then rinsed in ethanol (99.5%). Finally, the scaffolds werethoroughly dried under vacuum. The surface structure before andafter grafting was assessed by Fourier transform infrared (FTIR)spectrometry by freezing the porous discs in liquid nitrogen andcutting them through the middle into two slices of the same thick-ness. The outside surface as well as the interior pore surfaces, theinside, of the discs were then analyzed. A Perkin–Elmer Spectrum2000 FTIR, equipped with an attenuated total reflectance (ATR)crystal accessory (Golden Gate) was used, providing an analysisof the surface down to a depth of approximately 1 lm. All spectrawere the means of 32 individual scans in the 4000–600 cm�1

interval.

2.4. Coupling of resveratrol

The acid functionalized AA grafted porous scaffolds were cova-lently coupled to resveratrol according to the following protocol.Scaffolds were pre-wetted in water for 2 h. Resveratrol (1% w/v)and EDC (5% w/v) and ethanol (40 vol.%) were then added and al-lowed to react at room temperature for 24 h under gentle shaking.A hydrolysable ester linkage formed between resveratrol and theacid grafted PCL scaffolds. The scaffolds were rinsed with plentyof fresh distilled water and ethanol and vacuum dried. Scaffoldsthat underwent the same procedure but without resveratrol wereused as negative controls.

2.5. Cell culture

Bone marrow stromal cells were obtained from 6-week-oldmale Sprague–Dawley rats as described previously [10]. Briefly,the rats were killed using 4% isofluorane in CO2. The femurs wereaseptically excised and the bone marrow flushed out with 20 mlof a-MEM. After centrifugation at 1000 rpm for 5 min the cell pel-let was collected and cultured in a-MEM medium supplementedwith 10% fetal bovine serum (FBS), 1 mM L-glutamine and100 lg ml�1 gentamycin. Non-adherent cells were removed after24 h. After reaching 80% confluence the cells were seeded onto16 mm scaffolds, which were pre-soaked in culture medium forat least 24 h in 24-well plates at a density of 50,000 cells per well.Osteoblast differentiation was initiated the following day with cul-ture medium supplemented with 50 lg ml�1

L-ascorbic acid,10 mM b-glycerophosphate and 0.1 mM dexamethasone. The med-ium was changed twice per week. Based on our experience, cellsharvested by this procedure maintained their multipotent differen-tiation capacity [10].

2.6. Quantification of alkaline phosphatase (ALP) activity and ALPstaining

A Phosphatase Substrate Kit (Pierce, Rockford, IL) containingp-nitrophenyl phosphate disodium salt (pNPP) was used to quan-tify the ALP activity in the cell culture medium. The medium wascollected on days 3, 7, 14 and 21 and frozen at �80 �C until analy-sis. pNPP solution was prepared by dissolving two pNPP tablets in8 ml of distilled water and 2 ml of diethanolamine substrate buffer.About 50 ll of medium was taken from each sample and incubatedwith 100 ll well�1 pNPP solution for 30 min at room temperaturein a 96-well plate. About 50 ll of 2 N NaOH was then added to eachwell to stop the reaction. Unused culture medium treated in thesame way was used as a blank control. The absorbance was mea-sured at 405 nm in a kinetic ELISA reader (Spectra MAX 250,Molecular Devices, Sunnyvale, CA). A TRACP and ALP double stainkit (Kakara Bio Inc., Otsu, Japan) was used to stain for ALP in thecell–scaffold composites. The substrate for ALP was dissolved in10 ml of distilled water before use. After washing twice with

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phosphate-buffered saline (PBS) the cell–scaffold composites werefixed in fixation buffer for 5 min. After washing with distilled waterthree times, 1 ml of ALP substrate was added to each well. Sampleswere incubated at 37 �C for 30 min, followed by washing with dis-tilled water.

2.7. Staining for mineralization of the cell–scaffold construct

Rat bone marrow stromal cells were seeded on the scaffolds at50,000 cells per well in 24-well plates and cultured in bone-induc-ing medium for 4 weeks. Both alizarin red and von Kossa stainingwere used for analysis of in vitro mineralization. For von Kossastaining the cell–scaffold constructs were washed with PBS andfixed with 4% paraformaldehyde. After rinsing with distilled watereach scaffold was immersed in 2 ml of 5% silver nitrate and placedunder UV light for 30 min. To remove the unreacted silver the scaf-folds were subsequently immersed in 5% sodium thiosulfate for5 min, followed by rinsing with distilled water twice. For alizarinred staining the cell–scaffold constructs were fixed in ice-cold70% ethanol for 1 h and then incubated with 40 mM alizarin redat pH 4.2 for 10 min. Non-specific staining was removed by wash-ing twice with PBS.

2.8. Animal experiments

Ten male Sprague–Dawley rats (12 weeks, weighing �500 g, la-beled randomly 1–10) were used in the experiments in accordancewith Karolinska Institute Animal Care and Use Committee guide-lines (ethical approval number S 123-06). Critical calvarial bonedefects (5 mm in diameter) were created in the animals by the pro-cedure described by Bosch et al. [26]. Briefly, the rats were anes-thetized with 2.7 ml kg�1 i.p. Hypnorm/midazolam solution (onepart Hypnorm� (0.315 mg ml�1 fentanyl citrate and 10 mg ml�1

fluanisone) (Janssen Pharmaceuticals, Antwerp, Belgium) to twoparts distilled water and one part midazolam (5 mg/ml) (Dumex-Alpharma, Copenhagen, Denmark). Circular calvarial defects weremade on both parietal bones using a trephine burr without perfo-rating the dura mater. For rats 1–5 the left calvarial defects werefilled with porous PCL scaffolds without resveratrol incorporation(CTRL-PCL) and the right defects were filled with resveratrol-mod-ified PCL scaffolds (RSV-PCL). For rats 6–10 the left calvarial defectswere filled with RSV-PCL and the right defects were filled withCTRL-PCL. All animals were killed 8 weeks after surgery and thescaffolds together with surrounding bone were extracted fromthe skulls.

2.9. Digital radiography and data analysis

A Siemens Opti 150/30/50 HC-100 model 4803388 instrument(München, Germany) was used to acquire a plain digital radio-graph of the skull specimens. After acquisition the digital datawere blind analyzed by an observer using Sectra Bildvisare com-puter software (Sectra Imtec AB, Stockholm, Sweden). The areaand the mean X-ray density of the calvarial defects were recorded.

2.10. Histological study

The scaffolds from in vitro cell cultures and the specimens fromanimal experiments were rinsed with saline and fixed in 10% for-malin. The in vivo skull samples were decalcified in 10% formicacid for 2 weeks. To observe matrix formation inside the scaffoldsin the cell cultures 9 lm frozen sections were prepared from thecenter of the discs and stained with toluidine blue. For histologicalexamination of the calvarial implants the samples were embeddedin paraffin and 7 lm transverse sections cut at the center of thebone defects. Three histological sections, representing the center

of the original surgical defect, were stained with hematoxylinand eosin (H&E) and observed by light microscopy for histologicaland histomorphometric analyses. Computer-assisted histomor-phometry was performed to measure the amount of newly formedbone within the defect. The images of the histological sectionswere captured with a digital camera (Leica DFC490, Leica Micro-systems AB, Stockholm, Sweden) fitted to a light microscope (LeicaDMRBE) at an original magnification of 16�. The digital imageswere saved on a computer and histomorphometric analysis wascompleted using Leica Qwin Plus version 3.5.1. The criteria usedin this study to standardize the histomorphometric analysis ofthe digital images followed the work of Furlaneto et al. [27]. Thepercentage of newly formed bone was calculated and compared.

2.11. Immunohistochemistry (IHC)

Paraffin sections from the animal experiments were treated witha 0.3% H2O2 in methanol solution for 30 min at 37 �C. Then the sec-tions were incubated with 5% bovine serum albumin (BSA) and 2%normal goat serum (NGS) for 20 min to avoid non-specific immuno-reactions. Primary rabbit anti-bone sialoprotein (BSP) polyclonalantibody (AB1854, Chemicon, Stockholm, Sweden) diluted in PBScontaining 2% NGS and 5% BSA (1:2500) was applied for 24 h at4 �C. Biotinylated goat anti-rabbit IgG (AP132B, Chemicon) was usedas the secondary antibody (1:300 in PBS containing 2% NGS and 5%BSA). Finally, sections were treated with an ABC kit (Vector Labora-tories, Stockholm, Sweden) and immuno-positive loci were detectedby incubation with 3,30-diaminobenzidine tetrahydrochloride(DAB) solution (Vector Laboratories). Sections were counterstainedwith Mayer’s hematoxylin for 30 s.

3. Results

3.1. Linkage of resveratrol to acrylic acid grafted PCL scaffolds

PCL is one of the most popular resorbable candidates for bio-medical applications, with many favorable properties, being toughyet flexible, with documented non-toxicity. The number of carbox-ylic groups immobilized on the surfaces during grafting is directlyrelated to the grafting time and can be quantified as the mean sur-face number of COOH groups by titration [28]. After 15 min graft-ing of AA on PCL surfaces the COOH surface concentration was�2.4 lmol cm�2, constituting the maximum number of conjuga-tion points in subsequent coupling reactions. Spectra confirmedthe immobilization of AA chains on the surface. The typical C@Ostretching at 1721 cm�1 stemming from the ester functionality ofeach PCL repeating unit was, after grafting, mostly overlaid by anew peak emerging at 1705 cm�1 attributed to the C@O function-alitity of the carboxylic acid pendant groups in each repeating unitof AA. A hydroxyl band at 3000–3600 cm�1 was also present in thegrafted samples, as expected. The porous discs were cut in half toverify that the structure change mediated by grafting was presenton both the inner and the outer pore surfaces (Fig. 1). The carbox-ylic acid groups were then used as linkage groups in the covalentcoupling of resveratrol to the grafted surfaces. A water soluble car-bodiimide, EDC, was used as the coupling agent. The carboxylicacid groups on the graft chains reacted with the carbodiimideforming a reactive O-acylisourea intermediate that readily reactedwith hydroxyl moieties resulting in an ester bond between thegraft chains and resveratrol (Fig. 2). Two hydroxyl groups wereavailable on each resveratrol molecule, but coupling was expectedto be less likely to occur at the bisubstituted phenyl group of res-veratrol due to steric hindrance. The resveratrol was covalentlyconnected to the graft chain pendant carboxyl moieties via adegradable aliphatic ester linkage, providing a means for the resve-

Fig. 1. ATR-FTIR spectrum confirming the grafting of acrylic acid both on thesurface (c) and inside (b) the porous scaffold. (a) Non-grafted PCL. The C@Ostretching at 1721 cm�1 from the PCL repeating unit is overlaid by the peak fromthe C@O functionality of the carboxylic acid groups at 1705 cm�1 after grafting,seen as the shoulder in (b) and (c) on the 1721 cm�1 peak. As expected, the surfacehas a higher graft yield compared with the inside of the porous scaffold. A hydroxylband at 3000–3600 cm�1 is also present in the grafted samples, with the same trendas at �1700 cm�1.

Fig. 3. Incorporation of resveratrol in the porous PCL scaffolds significantlyincreased ALP activity of the co-cultured mesenchymal stem cells. The rat bonemarrow stromal cells were cultured on the RSV-PCL and CTRL-PCL scaffolds for upto 3 weeks. (A) ALP activity of the culture medium was measured by PNPP analysison days 3, 7, 14 and 21. The medium harvested from the RSV-PCL cell cultures hadsignificantly higher ALP activity compared with the medium from the CTRL-PCL cellcultures. (B) This effect could also be seen by ALP staining of the cell–polymercomposites with an ALP staining kit (Kakara Bio) on day 7. The experiment wasrepeated three times. Each data point represents the mean ± SEM of three samples.*P < 0.05 indicates significant difference to time matched CTRL-PCL.

754 Y. Li et al. / Acta Biomaterialia 7 (2011) 751–758

ratrol moiety to be released by hydrolytic cleavage during in vivouse of the scaffolds, hence providing a supply of the bioactive sub-stance in the host tissue.

3.2. In vitro study

ALP activity could be quantified in the conditional medium fromthe in vitro cultures by pNPP analysis. Fig. 3A shows a representa-tive result from one of the three independent measurements. Forcells seeded on control PCL the medium ALP activity increased dra-matically in the early culture period, reaching a peak on day 7(about 3.35 times the value on day 3). It then decreased, but re-mained at a constant level in the later culture period (about 2.02times the value on day 3 on day 14 and 1.98 times on day 21).For cells seeded on RSV-PCL a similar pattern of ALP activity wasfound, but at all three time points ALP activity was significantlyhigher compared with the control group (about 161.7% of thecontrol on day 7, 153.0% on day 14 and 130.2% on day 21). Consis-tent with the quantitative analysis, direct staining of intracellular

Fig. 2. Strategy for coupling resveratrol through a hydrolysable linkage to poly(epsilon-cbisubstituted phenyl group.

ALP on day 7 showed a much stronger signal on cells seeded onthe RSV-PCL scaffolds (Fig. 3B). In order to visualize the tissuestructure formed inside the scaffolds, frozen sections of the poly-mer–cell composites were stained with toluidine blue. As indicatedin Fig. 4A, more proteinaceous matrix was seen in the RSV-PCLscaffolds. Rat bone marrow stromal cells are known to form miner-alized nodules after long-term culture in bone-inducing medium,which could be shown by von Kossa and alizarin red staining[10]. We found that these methods could also be applied to thePCL–cell composites. Fig. 4B shows that after culture for 4 weeksthe RSV-PCL scaffolds demonstrated stronger staining.

3.3. In vivo study

All animals remained in good health and did not show anywound complications post surgery. The gross dimensions of the

aprolactone) surfaces. Reaction product B is less likely due to steric hindrance of the

Fig. 4. Incorporation of resveratrol in the porous PCL scaffolds increased matrixproduction and mineralization of the cell–scaffold co-cultures. The rat bone marrowstromal cells were cultured on the RSV-PCL and CTRL-PCL scaffolds for 4 weeks. (A)More matrix was seen in the RSV-PCL scaffolds on toluidine blue staining of thefrozen sections of the cell–scaffold composites. (B) von Kossa and alizarin redstaining showing stronger mineralization of the RSV-PCL scaffolds.

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original implants were still visible in the explanted calvaria. All im-plants were enveloped in thin tissue capsules without gross signsof inflammation or adverse tissue reaction, e.g. necrosis. The ratsdemonstrated individual variance in bone healing, which wasnoted by palpating the defect region at autopsy and confirmedby radiographic (Fig. 5) and histological (Fig. 6) analysis. Since eachrat was implanted with both types of scaffold a paired t-test wasused to analyze the difference in the mean X-ray density and defectsizes between RSV-PCL and CTRL-PCL implants. No significant dif-ference was found regarding the defect size between the two types

Fig. 5. Radiography of the rat calvarial defects 8 weeks after scaffold implantation.Two critical bone defects (5 mm in diameter) were created on both sides of theparietal bone in 10 male Sprague–Dawley rats. For rats 1–5 the left calvarial defectswere filled with CTRL-PCL scaffolds and the right defects with RSV-PCL scaffolds. Forrats 6–10 the scaffolds were placed in the opposite manner. All animals were killed8 weeks after surgery and the scaffolds together with the surrounding bone wereextracted from the skulls.

of implants. However, as shown in Table 1, the defects implantedwith RSV-PCL displayed a significantly higher X-ray density thanthe defects implanted with CTRL-PCL. This difference was furtherconfirmed by histological analysis. H&E staining showed bone-likeparticles inside the scaffolds. Fig. 6A–D show the representativehistological features from one of the experimental animals: thetwo scaffolds shared many common features, such as fibrousencapsulation, infiltration with elongated cells and collagen fibers(confirmed by type I collagen staining, data not shown), as well asbone extension from the defect edges. In the RSV-PCL scaffold anumber of bone-like structures were formed in the region nearthe dura mater, while similar structures could not be observed inthe CTRL-PCL scaffold. With the aid of computer-based histomor-phormetric analysis, the percentage of bone forming area wasquantified. Fig. 6E shows that RSV-PCL scaffolds had a significantlygreater area of bone regeneration than CTRL-PCL scaffolds (25.37%versus 10.57%, P < 0.01, paired t-test). In order to confirm bone for-mation in the bone-like structures the presence of a specific bonemarker, BSP, was assessed by IHC. As shown in Fig. 7, like the sur-rounding calvarial bone, the bone-like structures were positivelystained for BSP while the fibrous tissue inside the scaffolds wasnegative.

4. Discussion

Osteoconductive capacity is one of the major requirements forscaffolds used in bone tissue engineering. MSCs from the surround-ing tissue must proliferate and differentiate into osteoblasts andparticipate in bone regeneration. This process is activated andstimulated by a number of systemic and local factors which gener-ate a cascade of intracellular signals in the osteoprogenitors [29].Resveratrol has been shown to direct MSC differentiation towardsthe osteoblast lineage [10] and to stimulate the proliferation andactivity of pre-osteoblasts [7]. Resveratrol has a simple and stablestructure [30] and can be extracted from plants or industrially syn-thesized [6]. It has been documented that the physiologically mostactive form, i.e. trans-resveratrol, was stable for at least 28 days inbuffers ranging from pH 1 to 7 and no degradation was observedafter evaporation with alcohol [31]. These features, together withthe osteotrophic effects, suggest that resveratrol is a promisingcandidate for bone tissue engineering. In this study resveratrolwas incorporated into AA-functionalized porous PCL. PCL is consid-ered to be a non-toxic and tissue compatible polymer [16]. It is fre-quently used as a scaffold in bone tissue engineering because of itsresistance to rapid hydrolysis and good mechanical strength [32].

Our in vitro study showed that ALP activity in the conditionalmedium increased for up to 7 days and then decreased. ALP actsas a transmembrane receptor involved in osteoprogenitor–osteo-blast adhesion, migration and differentiation [33]. In vivo ALP ismainly detected in osteoprogenitors and pre-osteoblasts, well be-fore mineralization and prior to the expression of non-collagenousmatrix molecules [34,35]. The ALP expression pattern found in ourstudy is therefore consistent with the typical expression curve ofthis osteoblastic marker [36]. The relatively higher ALP activity inRSV-PCL cultures indicates that under our culture conditions theresveratrol released from PCL was biologically functional. Thegreater matrix formation inside the RSV-PCL discs, as demon-strated by toluidine blue staining, could be the result of eithermore osteoblasts derived from progenitor cells or enhanced matrixproduction. Like most MSC cultures, the cell–scaffold compositesshowed mineralization after culture in bone-inducing mediumfor 4 weeks. In vitro mineralization is known to be caused by co-precipitation of calcium (from the culture medium) and inorganicphosphates (generated by ALP catalyzed b-glycerophosphate deg-radation) onto a collagen matrix [37]. The stronger von Kossa

Fig. 6. H&E staining of the implanted polymers in rat 8. Both CTRL-PCL (A and C) and RSV-PCL (B and D) scaffolds were encapsulated by thin fibrous layers and filled with cellsand matrix. In RSV-PCL a number of bone-like structures (D) were seen extending from the dura side of the scaffold (B). However, these structures were not observed in CTRL-PCL. In addition, the cell number was much higher in CTRL-PCL, with many of the cells demonstrating a fibroblast phenotype. (A, B) 4� magnification; (C, D) 40�magnification. SS, subcutaneous side of the scaffold; DS, dura side of the scaffold; BLS, bone-like structures. (E) Histomorphometric analyses of bone regeneration in thecalvarial bone defects (P < 0.01, paired t-test).

Table 1X-ray analysis of bone regeneration.

Rat No. X-ray density (units)

CTRL RSV

1 1033 ± 53 1078 ± 772 1080 ± 54 1126 ± 973 1114 ± 64 1168 ± 694 980 ± 64 1048 ± 675 1092 ± 68 1135 ± 596 1093 ± 61 1174 ± 997 1027 ± 100 1346 ± 1158 1124 ± 50 1198 ± 879 1002 ± 58 1161 ± 9710 1044 ± 50 1111 ± 88Mean ± S.D. 1077 ± 66 1153 ± 82*P value P = 0.000251

* Paired-samples t-test was used for statistical analysis.

756 Y. Li et al. / Acta Biomaterialia 7 (2011) 751–758

and alizarin red staining of the RSV-PCL discs further supports theincreased ALP activity and matrix production. It should be pointed

out that we immersed the cell–scaffold composites in protein lysisbuffer in order to measure the expression of several intracellular ormembrane-bound osteoblast markers, such as Runx2, osterix, oste-ocalcin and osteopontin. However, there was too little protein toperform a successful Western blot. Despite this limitation, ourin vitro results provide a strong indication that incorporation ofresveratrol in porous PCL by vapor phase grafting can enhancethe bone-forming activity of osteoprogenitors.

To study the in vivo osteoinductive effects the polymers wereimplanted in rat calvarial defects for 8 weeks and bone regenera-tion was evaluated by X-ray and histological methods. We foundthat although the defects implanted with RSV-PCL scaffolds hadsignificantly higher X-ray density, their size was not significantlyreduced compared with CTRL-PCL implantation. We also noticedthat the main X-ray density difference between the two groupswas observed in the center rather than the edge of the scaffold.This finding differs from several previous reports in which boneregeneration originated from the edge of the calvarial defects[38,39]. The surrounding structures that possibly contribute to

Fig. 7. In order to confirm the new bone formation in RSV-PCL, a specific bonemarker, BSP was stained by IHC in paraffin sections. Like the surrounding calvarialbone (A), the bone-like structures in the RSV-PCL scaffold were positively stained byBSP (B), while the fibrous structures inside CTRL-PCL were negative (C). BLS, bone-like structures.

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the regeneration of calvarial defects include the periosteum, duraand adjacent bone [40]. Due to the much larger contact surface,the progenitor cells for skull morphogenesis and repair are consid-ered to be mainly located in the dural tissue [41,42]. This notionwas supported by our histological findings, which showed thatthe bone-like structures extended from the dura side. Since decal-cified samples are unsuitable for the study of mineralization, weevaluated the expression of BSP, a marker of terminally differenti-ated osteoblasts. BSP is selectively expressed in mineralizing tis-sues and, especially, at sites of de novo bone formation [43]. Asexpected, BSP could only be detected in the bone-like islands in-side all the implanted scaffolds and not in the surrounding fibrous

structures. Taken together, these results give a reasonable indica-tion that incorporation of resveratrol enhances the bone regenerat-ing capacity of porous PCL scaffolds. However, it has to be borne inmind that neither the increased X-ray density nor the enhancedbone island formation directly indicate better mechanical function,which could only be shown by biomechanical assay of the implantsand might be a task for future study.

Since its discovery resveratrol has been shown to have a num-ber of physiological properties that could be useful in human med-icine [11]. However, its rapid metabolism leads to an extremelylow plasma concentration of around 0–26 nM [44], which is sev-eral thousand fold lower than the concentrations demonstratingin vitro effects [10]. Therefore, combining resveratrol with biode-gradable polymers by vapor phase grafting may provide a newroute by which to deliver this agent to a local target and strengthenits potency. At present we have optimized neither the reagent con-centrations nor the amount of RSV needed to produce the optimaleffect. This could be one reason why bone regeneration in RSV-PCL,although significant compared with CTRL-PCL, was less than hasbeen shown for example on scaffolds containing BMPs [45]. There-fore, further studies, including a kinetic quantification of RSV re-leased from the scaffolds, enhancing the efficiency of resveratrolcoupling and/or the addition of other osteotrophic substances,are needed in order to optimize the bone regenerating effects. Fur-thermore, based on the broad range of health benefits of resvera-trol, we hope the advantage of this functionalization techniquecan be demonstrated for other disease models, such as cancers,diabetes and cardiovascular disease.

5. Conclusion

We have shown for the first time that incorporation of resvera-trol in vapor phase surface grafted AA-functionalized porous PCLsignificantly increased the osteoinductive ability of the scaffoldsboth in vitro and in vivo.

Acknowledgements

The study was supported by the program ‘‘Biomedical Func-tional materials” (Dnr: A3 02:139) funded by the Swedish Founda-tion for Strategic Research and the Ulla and Gustaf of UgglasFoundation. We thank Peter Plikk for helpful discussions aboutpolymer scaffolds.

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figures 3, 4, 6 and 7 aredifficult to interpret in black and white. The full colour images can befound in the on-line version, at doi:10.1016/j.actbio.2010.09.008.

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