Elsevier

Acta Biomaterialia

Volume 7, Issue 2, February 2011, Pages 751-758
Acta Biomaterialia

Resveratrol-conjugated poly-ε-caprolactone facilitates in vitro mineralization and in vivo bone regeneration

https://doi.org/10.1016/j.actbio.2010.09.008Get rights and content

Abstract

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

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.

References (45)

  • A.J. Salgado et al.

    Bone tissue engineering: state of the art and future trends

    Macromol Biosci

    (2004)
  • F.R. Rose et al.

    Delivery systems for bone growth factors – the new players in skeletal regeneration

    J Pharm Pharmacol

    (2004)
  • J.M. Wozney et al.

    Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair

    Clin Orthop Relat Res

    (1998)
  • L. Bavaresco

    Role of viticultural factors on stilbene concentrations of grapes and wine

    Drugs Exp Clin Res

    (2003)
  • L. Pirola et al.

    Resveratrol: one molecule, many targets

    IUBMB Life

    (2008)
  • K. Mizutani et al.

    Resveratrol attenuates ovariectomy-induced hypertension and bone loss in stroke-prone spontaneously hypertensive rats

    J Nutr Sci Vitaminol (Tokyo)

    (2000)
  • C.M. Backesjo et al.

    Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells

    J Bone Miner Res

    (2006)
  • J.A. Baur et al.

    Therapeutic potential of resveratrol: the in vivo evidence

    Nat Rev Drug Discov

    (2006)
  • Z.M. Huang et al.

    Encapsulating drugs in biodegradable ultrafine fibers through co-axial electrospinning

    J Biomed Mater Res A

    (2006)
  • S. Das et al.

    Formulation and optimization of zinc-pectinate beads for the controlled delivery of resveratrol

    AAPS Pharm Sci Tech

    (2010)
  • P.A. Gunatillake et al.

    Biodegradable synthetic polymers for tissue engineering

    Eur Cell Mater

    (2003)
  • N. Andronova et al.

    Resilient bioresorbable copolymers based on trimethylene carbonate, L-lactide, and 1,5-dioxepan-2-one

    Biomacromolecules

    (2006)
  • Cited by (72)

    • Functional improvement of collagen-based bioscaffold to enhance periodontal-defect healing via combination with dietary antioxidant and COMP-angiopoietin 1

      2022, Biomaterials Advances
      Citation Excerpt :

      Unlike rhCOMP-Ang1, coumaric acid is a naturally occurring small-molecule phenolic acid that exhibits antioxidant, anti-inflammatory, anti-ulcer, and anti-diabetic properties [12–16]. The antioxidant property of natural compounds is important in scavenging reactive oxidants and suppressing inflammatory responses that also stimulate osteoclastic activation [9,14,16,41]. Together, this suggests that rhCOMP-Ang1 released from an ACS initially and rapidly stimulates angiogenic and osteogenic responses accompanied by migration of osteogenic progenitor cells around the defects, where coumaric acid maintains a cellular redox state and inhibits inflammatory responses and bone resorption.

    • Current natural bioactive materials in bone and tooth regeneration in dentistry: a comprehensive overview

      2021, Journal of Materials Research and Technology
      Citation Excerpt :

      In an animal study intraperitoneal injection of resveratrol (10 μmol/kg) significantly improved bone regeneration process after dental extraction [204]. In pre-clinical settings many studies have evaluated bone and cartilage regenerative ability of tissue scaffolds such as collagen [205,206], chitosan [207], poly-ε-caprolactone [208], Polycaprolactone [209] and HA [210] after enriching them with resveratrol. The majority of the results were promising.

    View all citing articles on Scopus
    View full text