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Research Papers

Cellular Response to Cyclic Compression of Tissue Engineered Intervertebral Disk Constructs Composed of Electrospun Polycaprolactone

[+] Author and Article Information
Andrea Fotticchia

Mechanical, Electrical, and Manufacturing
Engineering Department,
Loughborough University,
Loughborough LE11 3TU, UK
e-mail: a.fotticchia@lboro.ac.uk

Emrah Demirci

Mechanical, Electrical, and Manufacturing
Engineering Department,
Loughborough University,
Loughborough LE11 3TU, UK
e-mail: e.demirci@lboro.ac.uk

Cristina Lenardi

Fondazione Filarete and CIMaINa,
Dipartimento di Fisica,
Universita' di Milano,
Via Celoria 16,
Milano 20133, Italy
e-mail: cristina.lenardi@mi.infn.it

Yang Liu

Mechanical, Electrical, and Manufacturing
Engineering Department,
Loughborough University,
Loughborough LE11 3TU, UK
e-mail: y.liu3@lboro.ac.uk

1Corresponding author.

Manuscript received August 18, 2017; final manuscript received February 2, 2018; published online March 16, 2018. Assoc. Editor: Carlijn V.C Bouten.

J Biomech Eng 140(6), 061002 (Mar 16, 2018) (9 pages) Paper No: BIO-17-1372; doi: 10.1115/1.4039307 History: Received August 18, 2017; Revised February 02, 2018

There is lack of investigation capturing the complex mechanical interaction of tissue-engineered intervertebral disk (IVD) constructs in physiologically relevant environmental conditions. In this study, mechanical characterization of anisotropic electrospinning (ES) substrates made of polycaprolactone (PCL) was carried out in wet and dry conditions and viability of human bone marrow derived mesenchymal stem cells (hMSCs) seeded within double layers of ES PCL were also studied. Cyclic compression of IVD-like constructs composed of an agarose core confined by ES PCL double layers was implemented using a bioreactor and the cellular response to the mechanical stimulation was evaluated. Tensile tests showed decrease of elastic modulus of ES PCL as the angle of stretching increased, and at 60 deg stretching angle in wet, the maximum ultimate tensile strength (UTS) was observed. Based on the configuration of IVD-like constructs, the calculated circumferential stress experienced by the ES PCL double layers was 40 times of the vertical compressive stress. Confined compression of IVD-like constructs at 5% and 10% displacement dramatically reduced cell viability, particularly at 10%, although cell presence in small and isolated area can still be observed after mechanical conditioning. Hence, material mechanical properties of tissue-engineered scaffolds, composed of fibril structure of polymer with low melting point, are affected by the testing condition. Circumferential stress induced by axial compressive stimulation, conveyed to the ES PCL double layer wrapped around an agarose core, can affect the viability of cells seeded at the interface, depending on the mechanical configuration and magnitude of the load.

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Figures

Grahic Jump Location
Fig. 1

Schematics representing the angle-ply configuration of double layers and the preparation for fluorescence microscopy analysis (a). Cell seeding strategy of the ES PCL matrix prior to overlaying with the top layer for the IVD-like construct preparation (b) and the structure of the final construct (c). Timeline of cell seeding, loading on and retrieving from bioreactor for test (d).

Grahic Jump Location
Fig. 2

(a) and (b) Representative SEM images of aligned fibers with 0.46±0.14 (a) and 1.72±0.50 μm, (b) fiber diameter. Histograms represent the angle distribution of ES PCL fibers. Scale bar 20 μm, (c)–(e) hMSCs attached to aligned fibers arranged into monolayer (c) and double layer, bottom (d) and top (e) layer. Cell nuclei are stained blue and actin filaments red. Scale bar 100 μm.

Grahic Jump Location
Fig. 3

Mechanical properties of aligned ES PCL fibers stretched at different angles in dry and wet conditions. (a) Elastic modulus, (b) strain at ultimate tensile strength, and (c) ultimate tensile strength. Note: asterisks indicate significant difference (p < 0.05) between dry and wet conditions within the same stretching angle.

Grahic Jump Location
Fig. 4

(a) Viability of cells seeded on double layers of different fiber diameters and (b) viability of cells seeded on double layers characterized by different thicknesses. Black bars on the right-hand side indicate significant difference (p < 0.05).

Grahic Jump Location
Fig. 5

(a) Agarose disk surrounded by an AF-like double-layer strip. The protruding edges labeled with red circle were used for handling the specimen during the sealant process and for loading onto the bioreactor. (b) Representative S-S curves of agarose disk and agarose disk surrounded by ES PCL layers. (c) Calculated circumferential stress experienced by the double-layer ES PCL layers from the average of three samples.

Grahic Jump Location
Fig. 6

(a) Schematic diagram showing the translation of compressive stress to circumferential stress experienced by the double layers of ES PCL with cells presented at the interface. (b) Viability of 5% mechanically stimulated IVD-like constructs. The assay was performed on both static and stimulated samples, at the same time points, before and after applying mechanical stimulation. Bars indicate p < 0.05 significance. (c) Representative images acquired at the end of mechanical stimulation experiments. Inner layer is the one in contact with the agarose gel and cells facing outward; the outer layer wraps both the inner layer and the agarose core with cells facing inward. Scale bar 100 μm.

Grahic Jump Location
Fig. 7

(a) Schematic diagram showing the translation of compressive stress by the configuration of the IVD-like constructs and (b) impaired cell attachment and viability as a consequence of fiber stretching and rotation in response to biaxial tensile stress (cells labeled blue represent those cells with compromised viability)

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