Resveratrol-conjugated poly-ε-caprolactone facilitates in vitro mineralization and in vivo bone regeneration
Introduction
A plethora of local growth factors have been identified with osteotrophic properties, such as bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs) and insulin-like growth factors I and II (IGF I/II) [1]. In bone tissue engineering these factors have been combined with different materials, ranging from inorganic bone graft substitutes (e.g. hydroxyapatite, calcium phosphate-based cements) and natural tissue components (e.g. collagen, hyaluronan) to different types of synthetic polymers, with the purpose of creating osteoinductive or osteoconductive scaffolds [2]. Although promising results have been noted, these factors are relatively unstable during industrial processing, making their application costly and the techniques demanding [3]. Therefore, the search for other osteotrophic substances has become an intriguing route to the 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 a variety of health benefits in mammals, among which the cancer chemopreventive and cardio- and neuro-protective properties have attracted most attention [6]. Its osteotrophic effects were first reported 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 be blocked by tamoxifen, an anti-estrogen, they concluded that resveratrol acted via estrogen receptors (ER) [7]. This notion was substantiated by their subsequent in vivo study, which showed that resveratrol prevented femoral bone loss in ovariectomized (OVX) rats [8]. A recent study by Su et al. [9] further clarified the underlying mechanism of resveratrol-mediated bone protection. They found that resveratrol stimulated BMP-2 production by osteoblasts through Src kinase-dependent ER activation and increased the serum concentration of BMP-2 in OVX rats [9]. Besides the ER/BMP-2 pathway, our previous findings indicated that resveratrol epigenetically modified the gene expression in mesenchymal stem cells (MSCs) through activation of Sirt1, a NAD-dependent histone deacetylase, which resulted in increased osteoblast differentiation in 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 bioavailability [11]. Recently, attempts at developing synthetic carrier systems for target-specific delivery of resveratrol have been reported [12], [13], [14], [15], however, their effects have not been evaluated using in vivo models. Synthetic biodegradable polymers are often used as protein carriers since their structure, geometry, porosity, mechanical properties, surface properties and degradation kinetics can be tailored according to requirements [16], [17], [18], [19], [20]. We have previously invented a vapor phase grafting method for surface functionalization of synthetic polymers in a non-degrading, solvent-free fashion [21]. This method for covalent immobilization of a hydrophilic graft chain on the substrate surface has been shown to not only significantly increase hydrophilicity and biocompatibility of the material [21], [22], [23] but also facilitate the attachment of bioactive molecules [24]. In the present study we functionalized the inner and outer surfaces of a porous poly-ε-caprolactone (PCL) scaffold by vapor phase grafting with acrylic acid (AA). The graft chain carboxylic acid pendant groups were then covalently bonded with resveratrol through a hydrolysable linkage. The biocompatibility and osteoconductive effect of this new scaffold were evaluated in rat bone marrow stromal cell cultures and rat calvarial defect models.
Section snippets
Reagents
Modified Eagle’s medium alpha (α-MEM), fetal bovine serum (FBS), l-glutamine and gentamycin were purchased from Invitrogen (Life Technologies, Paisley, UK). Resveratrol was purchased from Sigma–Aldrich. Polymer scaffolds were produced from poly-ε-caprolactone (PCL) (Mn = 80000, Aldrich) using chloroform (Labscan) and NaCl (Fischer, ground and dried before use). The polymer structure and molecular weight was verified by 1H NMR and size exclusion chromatography (SEC) analyses. Benzophenone (BPO)
Linkage of resveratrol to acrylic acid grafted PCL scaffolds
PCL is one of the most popular resorbable candidates for biomedical applications, with many favorable properties, being tough yet flexible, with documented non-toxicity. The number of carboxylic groups immobilized on the surfaces during grafting is directly related to the grafting time and can be quantified as the mean surface number of COOH groups by titration [28]. After 15 min grafting of AA on PCL surfaces the COOH surface concentration was ∼2.4 μmol cm−2, constituting the maximum number of
Discussion
Osteoconductive capacity is one of the major requirements for scaffolds used in bone tissue engineering. MSCs from the surrounding tissue must proliferate and differentiate into osteoblasts and participate in bone regeneration. This process is activated and stimulated by a number of systemic and local factors which generate a cascade of intracellular signals in the osteoprogenitors [29]. Resveratrol has been shown to direct MSC differentiation towards the osteoblast lineage [10] and to
Conclusion
We have shown for the first time that incorporation of resveratrol in vapor phase surface grafted AA-functionalized porous PCL significantly increased the osteoinductive ability of the scaffolds both in vitro and in vivo.
Acknowledgements
The study was supported by the program “Biomedical Functional materials” (Dnr: A3 02:139) funded by the Swedish Foundation for Strategic Research and the Ulla and Gustaf of Ugglas Foundation. We thank Peter Plikk for helpful discussions about polymer scaffolds.
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