CCP マスキュラーコレクション 匠シリーズ 悪魔将軍オプション 虹彩箔

Fibrous scaffolds made by electrospinning for
tendon repair
Lucy A Bosworth
School of Materials, The University of Manchester,
Oxford Road, Manchester, M13 9PL
lucy.bosworth@manchester.ac.uk
The biomaterials and tissue engineering sector has become a routinely accepted
practice for improving people’s quality of life – be it contact lenses, hip and knee
replacements, or skin grafts. Coupled with an ageing population there is an
increasing need for regenerative medicines.
The majority of biomaterial and tissue engineering applications – regardless of their
final location in the body – require the fabrication of a construct, with dimensions and
structure that can fill the damaged area, promote tissue ingrowth and perform the
original tissues role. Constructs, more commonly referred to as scaffolds, often aim
to replicate the original tissues ultra-structure.
Scaffolds fabricated via electrospinning are being increasingly used in a wide range
of biomaterial applications due to the ability to create nanoscopic fibres with
diameters as small as 5 nm [1]. Additionally, the flexibility of this technique allows
networks of nanofibres to be created in many different orientations and sizes that are
comparable to the ultra-structures of many tissues [2].
An inexpensive and relatively easy set-up, electrospinning uses high voltages to
generate internal repulsive forces within a polymer solution, which are then expelled
in the form of fine diameter fibres. In brief, a voltage supply is connected to a
needle-tipped syringe containing a flowing solution of polymer dissolved in solvent.
An earthed, target collector is positioned at a set distance from the needle.
Application of a high voltage causes charging of the polymeric solution and formation
of a Taylor cone [3]. Once the charge intensity of the solution is sufficient to
overcome the surface tension and visco-elastic forces of the Taylor cone, the
polymer is expelled as a charged jet [4]. This jet travels across an air-gap whilst
simultaneously undergoing solvent evaporation and stretching and thinning of the
polymer occurs due to charge repulsion before coming into contact with the earthed
collector, at which point the process is complete, charges dissipate and a network of
fibres are formed.
The last 8 years of research has been spent focusing on utilising this technique to
produce electrospun scaffolds as a potential new therapy for repairing damaged
tendons. In order to achieve this, it is important to understand the tissue to be
repaired/replaced and the clinical need. Tendons are a type of connective tissue that
frequently withstand high tensile loads, which can predispose them to injuries (or
tendinopathies), such as, spontaneous rupture or chronic degeneration of the tissue
leading to eventual rupture. They are also susceptible to other types of injuries,
including lacerations. With a poor healing response, there are a number of surgical
interventions available that aim to restore the tendon to its original structure and
function; yet there is no clear leader and autografting remains the gold standard
therapy for segmental repairs or reconstructions. However, problems can arise as
there is no guarantee the patient will have sufficient, usable tendon tissue that can be
sourced from a secondary site. Additionally when healthy tissue can be harvested,
the patient is left susceptible to an increased risk of infection and prolonged
rehabilitation. Predominantly composed of uniaxially aligned collagen fibres that take
on a hierarchical structure, electrospinning lends itself as the ideal technique for
producing fibrous scaffolds that can imitate the natural tendon tissue [5].
By electrospinning onto the edge of a mandrel rotating at an optimised speed, highly
aligned fibres can be collected [6]. As shown in the schematic, these ribbons of
aligned fibres can be further manipulated to create 3D bundles of fibrous yarn that
are more reminiscent of the tendon hierarchy than the initial 2D electrospun sheet of
fibres (Figure 1). In addition to resembling the natural tendon structure, there are
several other key factors that should be incorporated when developing synthetic
scaffolds for this type (and many other tissues) – the scaffold should (i) be
biocompatible and promote an appropriate tissue healing response, (ii) provide
sufficient mechanical strength to withstand applied forces and support new tissue
formation, and (iii) degrade with time without build-up of toxic by-products and at a
rate that allows a smooth load transfer.
Figure 1 – Electrospinning set-up and process for fabricating 2D fibrous ribbons and formation of 3D
yarns.
Initially a range of polymers and solvents were trialled and it was possible to create
yarns from each combination [7].
However, their tensile properties varied
significantly: use of poly(lactic-co-glycolic) acid (PLGA85:15) resulted in greatest
stiffness, but did not have a correspondingly high tensile strength. Similarly use of
acetone as a solvent resulted in both low stiffness and strength.
Poly(εcaprolactone) (PCL) dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) was
deemed to be the optimal polymer/solvent combination for this research, providing
Young’s Modulus and tensile strength of 68 MPa and 42 MPa, respectively.
In vitro characterisation of PCL/HFIP yarns were found to support the adhesion and
proliferation of both equine tenocytes and human mesenchymal stem cells (hMSC)
(Figure 2a-d). In addition, the surface topography of the scaffold influenced cell
behaviour causing cells to align parallel to the underlying fibres – similar to their
natural morphology within tendon tissue [6]. Analysis of gene expression for hMSCseeded yarns subjected to a tensile, sinusoidal loading regime for one hour each day
for 21 days, suggested their differentiation towards a tendon-lineage as an upregulation was observed in several key genes, including; collagen type I and III,
tenascin-C, elastin and fibronectin [8] (Figure 2e).
Figure 2 - Scanning Electron Micrographs of poly(ε-caprolactone) electrospun fibres with
equine tenocytes after 1 week (A) and 2 weeks (B), and human Mesenchymal stem cells after
1 week (C) and 2 weeks (D); (E) Gene expression markers for hMSC-seeded scaffolds (n=4)
subjected to cyclic loading compared to hMSC-seeded scaffolds held under static conditions.
(Data represented as mean ± st. deviation; T-test - ** p<0.01, * p<0.001)
In vivo assessment of electrospun yarns implanted into critical sized defects within
the flexor digitorum profundus tendons of mice was performed over a six-week time
frame (Figure 3). Haematoxylin and Eosin (H&E) staining after three days showed
the outer perimeter of the PCL graft to be completely surrounded by a thick layer of
cells. By three weeks, cells had migrated through to the scaffold core, which is
essential for integration and the long-term success of the implant. Yet by six weeks,
the PCL material appeared to have undergone complete degradation and a
prominent cell response was observed.
Comparison to autograft similarly
demonstrated a high cell presence at six weeks, which could be due to the tissue
remodelling.
Tendons are known to be slow-healing tissues and as such,
degradation of the PCL at this rate is too fast. To overcome this, we are in the
process of repeating these animal studies using a different grade of PCL and our
initial observations demonstrate this material to still be present and intact at six
months.
Figure 3 - H&E light microscopy images of poly(ε-caprolactone) scaffolds and autograft tissue
within murine tendons at 3 days, 3 weeks and 6 weeks. Arrows indicate position of graft;
scale bar = 500 µm.
Currently funded by the MRC-DPFS, the project team is continuing to make
significant progress in the development of this scaffold, and is focusing on up-scaling
this prototype as a device for clinical translation and use in patients with damaged
tendons.
Acknowledgements
With special thanks to Dr Sarah Cartmell and Prof Sandra Downes for guidance and
supervision; and Prof Duncan McGrouther, Dr Jason Wong and Dr Sarah Rathbone
and for scientific input. Thanks also to the EPSRC, UMIP Premier Fund, RegeNer8
and MRC-DPFS for financial support.
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