Elsevier

Biomaterials

Volume 149, December 2017, Pages 51-62
Biomaterials

Three-dimensional piezoelectric fibrous scaffolds selectively promote mesenchymal stem cell differentiation

https://doi.org/10.1016/j.biomaterials.2017.09.024Get rights and content

Abstract

The discovery of electric fields in biological tissues has led to efforts in developing technologies utilizing electrical stimulation for therapeutic applications. Native tissues, such as cartilage and bone, exhibit piezoelectric behavior, wherein electrical activity can be generated due to mechanical deformation. Yet, the use of piezoelectric materials have largely been unexplored as a potential strategy in tissue engineering, wherein a piezoelectric biomaterial acts as a scaffold to promote cell behavior and the formation of large tissues. Here we show, for the first time, that piezoelectric materials can be fabricated into flexible, three-dimensional fibrous scaffolds and can be used to stimulate human mesenchymal stem cell differentiation and corresponding extracellular matrix/tissue formation in physiological loading conditions. Piezoelectric scaffolds that exhibit low voltage output, or streaming potential, promoted chondrogenic differentiation and piezoelectric scaffolds with a high voltage output promoted osteogenic differentiation. Electromechanical stimulus promoted greater differentiation than mechanical loading alone. Results demonstrate the additive effect of electromechanical stimulus on stem cell differentiation, which is an important design consideration for tissue engineering scaffolds. Piezoelectric, smart materials are attractive as scaffolds for regenerative medicine strategies due to their inherent electrical properties without the need for external power sources for electrical stimulation.

Introduction

Tissue engineering or regenerative medicine offers a promising approach to repair damaged tissues by combining cells with biomaterials that act as scaffolds to facilitate tissue growth. The biomaterial can be designed to mimic the native tissue extracellular matrix providing appropriate cues for desired cell function. Endogenous electrical fields have been well established during embryonic development, wound healing and limb regeneration (Reviewed in Ref. [1]). The electrical activity generated can be associated with extracellular matrix materials, such as collagens [2] and glycosaminoglycans (GAGs) [3], which display piezoelectric activity. Specifically, they are capable of converting mechanical strain into electrical output. Tissues, such as bone and cartilage, which contain these materials, have been known to display electrical behavior when subjected to loading or deformation [4], [5], [6]. Yet, this phenomena of piezoelectricity has largely been unexplored as a potential scaffold strategy in the tissue engineering field (Reviewed in Refs. [7], [8]).

The development of smart materials for biological and biomedical applications is an emerging field. Combining biological entities such as DNA, cells or tissues with soft or flexible piezoelectric materials can yield devices that can dynamically sense and adapt to environmental cues with or without the use of external stimuli. Yet flexible, piezoelectric biomaterials have only been in the form of thin-films, tubes, non-woven or aligned fiber membranes, and isolated fibers (Reviewed in Refs. [7], [8]), which limits their use for tissue regeneration applications or as in vitro tissue models. For the growth of three-dimensional (3-D) tissues, we have fabricated 3-D piezoelectric fibrous scaffolds made of poly(vinylidene fluoride – trifluoroethylene) (PVDF-TrFE), which displays the greatest piezoelectric activity of known polymers [9], [10], are biocompatible [11], [12] and have been shown to stimulate cell function in a variety of cell types [12], [13], [14], [15], [16], [17], [18], [19]. Previous studies have reported the fabrication of electrospun PVDF-TrFE fiber based nanogenerators and characterized their piezoelectric properties under compressive loads or displacements [20], [21], making them suitable for self-power generators for energy harvesting applications. This level of output also may induce biological activity. The advantage of using PVDF-TrFE fibrous scaffolds is that externally applied electrodes are not needed wherein electrical stimulation can be generated through physiological movement.

In this study, piezoelectric PVDF-TrFE fibrous scaffolds were evaluated for promoting stem cell differentiation and in vitro tissue growth as a first study in demonstrating the potential of 3-D flexible smart materials as tissue engineering scaffolds. PVDF-TrFE scaffolds were either electrospun (as-spun) or electrospun and subsequently heat-treated (annealed) to increase the polar/piezoelectric β-phase crystal content and piezoelectric properties. Mesenchymal stem cell (MSC) differentiation towards chondrogenic and osteogenic lineages on these scaffolds was examined in a dynamic bioreactor where cyclic compression was applied at a physiological frequency, activating the piezoelectric properties of the scaffold. Polycaprolactone (PCL), a non-piezoelectric control, was used since PVDF-TrFE cannot be processed into a non-piezoelectric form due to its piezoelectric β-phase content. Although PCL has a different surface chemistry from PVDF-TrFE, PCL was chosen primarily due to its slow degradation rate and ease in fabricating fibrous scaffolds similar in morphology and size to PVDF-TrFE scaffolds. Additionally, PCL is well known for its biocompatibility with many cell types and is in clinical use [22]. We hypothesized that piezoelectric scaffolds undergoing dynamic compression will enhance chondrogenesis and osteogenesis and in vitro tissue formation. Findings demonstrated that MSC differentiation was dependent upon the level of piezoelectric activity of the scaffold, where lower levels promoted chondrogenesis and higher levels promoted osteogenesis. Piezoelectric activity promoted greater differentiation as noted by both matrix synthesis and gene expression than mechanical loading alone, demonstrating the effect of electromechanical stimulation on MSC differentiation.

Section snippets

3-D piezoelectric scaffold fabrication and characterization

3-D scaffolds were prepared using the electrospinning technique. Electrospinning is a dynamic process where an electric field is applied to an ejecting polymer solution resulting in the formation of fibers that collect on a grounded plate. Conventional electrospinning has the limitation of producing two-dimensional (2-D) sheets or membranes however, we were able to fabricate thick, continuous electrospun scaffolds (Fig. 1A) using a two power supply setup, in contrast to the commonly used one

Discussion

Piezoelectric, 3-D fibrous PVDF-TrFE scaffolds were evaluated for MSC chondrogenesis and osteogenesis under dynamic loading conditions, where the processing of the scaffolds impacted the level of piezoelectric activity which influenced differentiation. The results demonstrated an increased chondrogenic differentiation of MSCs on as-spun PVDF-TrFE scaffolds, which have lower piezoelectric activity, as determined from GAG, collagen type II to I ratio and gene expression. Additionally, annealed

Conclusions

Piezoelectric materials hold promise as a scaffold strategy to enhance tissue formation by providing a smart, electrically active microenvironment without the use of an external power source. Here, we show that piezoelectric materials can be fabricated into flexible, three-dimensional fibrous scaffolds that can be used to stimulate mesenchymal stem cell differentiation and tissue formation when undergoing dynamic loading, which mimics physiological loading conditions found in structural

Materials and methods

Scaffold Fabrication: Fabrication of fibrous scaffolds was accomplished using electrospinning technique. The electrospinning setup consisted of solution of 25 wt/vol% poly (vinylidene difluoride – trifluoroethylene) (65/35, PVDF-TrFE, Solvay Solexis) solution in methyl ethyl ketone (Fisher Scientific). The polymer solution was transferred to a 10 mL syringe fitted with a stainless steel needle. The syringe was placed on a syringe pump (Harvard Apparatus) with a set flow rate of 15 mL/h and a

Biological studies

Scaffold Sterilization: All scaffolds were cut into 6 mm diameter disks using a biopsy punch (Miltek, PA, USA). The thickness of scaffold with ∼3 mm were chosen for biological studies. The scaffolds were sterilized with 100% ethanol (Fisher Scientific, USA) for 20 min and later air dried in sterile hood overnight prior to cell seeding.

Human Mesenchymal Stem Cells (MSCs) Isolation and Culture: Human MSCs were isolated from whole bone marrow aspirates (Lonza Biosciences, MD), which were collected

Acknowledgements

The authors would like to thank Martin Garon at Biomomentum, Inc. for the use of their Mach-1 tester to collect streaming potential data of the piezoelectric scaffolds. The authors would like to thank funding support from the National Science Foundation (1006510 and 1610125), National Key Research and Development Program of China (2016YFA0201001) and Science and Technology Plan of Shenzhen City (JCYJ20160331191436180), and the National Science Foundation - Science and Technology Center – Center

References (66)

  • D.R. Eyre et al.

    Covalent cross-linking of the NC1 domain of collagen type IX to collagen type II in cartilage

    J. Biol. Chem.

    (2004)
  • F. Barry et al.

    Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components

    Exp. Cell Res.

    (2001)
  • J. Xu et al.

    Signal transduction in electrically stimulated articular chondrocytes involves translocation of extracellular calcium through voltage-gated channels

    Osteoarthr. Cartil.

    (2009)
  • H.R. Pant et al.

    Fabrication of highly porous poly (ɛ-caprolactone) fibers for novel tissue scaffold via water-bath electrospinning

    Colloids Surf. B Biointerfaces

    (2011)
  • S. Haynesworth et al.

    Characterization of cells with osteogenic potential from human marrow

    Bone

    (1992)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method

    Methods

    (2001)
  • M.A. Messerli et al.

    Extracellular electric fields direct wound healing and regeneration

    Biol. Bull.

    (2011)
  • M. Minary-Jolandan et al.

    Nanoscale characterization of isolated individual type I collagen fibrils: polarization and piezoelectricity

    Nanotechnology

    (2009)
  • C.A. Bassett et al.

    Electrical behavior of cartilage during loading

    Science

    (1972)
  • M.A. Brady et al.

    The application of multiple biophysical cues to engineer functional neocartilage for treatment of osteoarthritis (part I: cellular response)

    Tissue Eng. Part B Rev.

    (2014)
  • B.M. Isaacson et al.

    Bone bioelectricity: what have we learned in the past 160 years?

    J. Biomed. Mater. Res. Part A

    (2010)
  • V.C. Mow et al.

    Mechano-electrochemical properties of articular cartilage: their inhomogeneities and anisotropies

    Annu. Rev. Biomed. Eng.

    (2002)
  • A.J. Lovinger

    Ferroelectric polymers

    Science

    (1983)
  • H. Ohigashi et al.

    Piezoelectric and ferroelectric properties of P (VDF-TrFE) copolymers and their application to ultrasonic transducers

    Ferroelectrics

    (1984)
  • Y.S. Lee et al.

    The influence of piezoelectric scaffolds on neural differentiation of human neural stem/progenitors cells

    Tissue Eng. Part A

    (2012)
  • G.G. Genchi et al.

    P (VDF-TrFE)/BaTiO3 nanoparticle composite films mediate piezoelectric stimulation and promote differentiation of SH-SY5Y neuroblastoma cells

    Adv. Healthc. Mater.

    (2016)
  • Genchi et al.

    P (VDF-TrFE)/BaTiO3 Nanoparticle Composite Films Mediate Piezoelectric Stimulation and Promote Differentiation of SH-SY5Y Neuroblastoma Cells

    Advanced healthcare materials

    (2016)
  • R. Augustine et al.

    Electrospun poly (vinylidene fluoride-trifluoroethylene)/zinc oxide nanocomposite tissue engineering scaffolds with enhanced cell adhesion and blood vessel formation

    Nano Res.

    (2017)
  • P. Hitscherich et al.

    The effect of PVDF-TrFE scaffolds on stem cell derived cardiovascular cells

    Biotechnol. Bioeng.

    (2016)
  • L. Persano et al.

    High performance piezoelectric devices based on aligned arrays of nanofibers of poly (vinylidenefluoride-co-trifluoroethylene)

    Nat. Commun.

    (2013)
  • K. Lau et al.

    Effect of annealing temperature on the morphology and piezoresponse characterisation of poly (vinylidene fluoride-trifluoroethylene) films via scanning probe microscopy

    Adv. Condens. Matter Phys.

    (2013)
  • D. Mao et al.

    Ferroelectric properties and polarization switching kinetic of poly (vinylidene fluoride-trifluoroethylene) copolymer

    Ferroelectr. - Phys. Eff.

    (2011)
  • M. Baniasadi et al.

    Thermo-electromechanical behavior of piezoelectric nanofibers

    ACS Appl. Mater. Interfaces

    (2016)
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