(155f) Nanofiber Scaffolds Mimicking Structural Organization of Tendon-to-Bone Insertion Site

Nanofiber Scaffolds Mimicking Structural
Organization of

Tendon-to-Bone Insertion Site

Jingwei
Xie^1*, Bing Ma¹,
Shuler D. Franklin²

¹Marshall
Institute for Interdisciplinary Research and Center for Diagnostic Nanosystems,
Marshall University, Huntington, WV, 25755 USA

²Department
of Orthopaedic Surgery, Joan C. Edwards School of Medicine, Marshall University,
Huntington, WV 25701 USA

^*Correspondence
should be addressed to: xiej@marshall.edu

Introduction

It is known that tendons are attached to
bones across a specialized transitional tissue with varying structures (i.e.,
collagen fibers are less oriented at the insertion compared to the tendon) and
compositions (i.e., the relative mineral concentration vs. distance across the
insertion site shows an approximately linear increase across the interface.¹
Efforts have been devoted to the development of scaffolds which could mimic the
structure (i.e., collagen fiber orientation) and/or composition (i.e., mineral
content) of tendon-to-bone insertions for the repair of rotator cuff injury. A
number of studies demonstrated the fabrication of scaffolds with a gradient of
mineral content for mimicking the composition and mechanical function of the
interface between soft tissue to hard tissue (i.e., tendon-to-bone,
ligament-to-bone, and cartilage-to-bone).^2-4 However, few studies
have centered on the creation of scaffolds capable of exhibiting the unique
structural organization of collagen fibers seen at the native tendon-to-bone
insertion site except that a recent study attempted to fabricate
?aligned-to-random? nanofiber scaffolds by making use of a specially-designed
collector during electrospinning for mimicking the structure of the
tendon-to-bone insertion site.⁵ But some issues still remain in this
study: the nanofiber scaffold has no gradual transitions from aligned portion
to random portion; and there is apparent difference of the thicknesses between
the two portions, which could easily cause breaking at the interface upon
exerting a force. Therefore, so far no such a scaffold can fully recreate the
gradients in structure found at the uninjured tendon-to-bone attachment.

In this study, we report a new and
simple method for fabrication of nanofiber scaffolds with gradations in fiber
organization by depositing random fibers on the uniaxially-aligned nanofiber mat
in a gradient manner. These surfaces mimic the change in fiber orientation
which presents at the tendon-to-bone insertion site. Tailoring the surface
features in a user-specified manner can provide spatial control over cell
response like cell morphology. Hence, we hypothesize that adipose-derived stem
cells (ADCSs) could respond differently to different locations of such
nanofiber surfaces. An adipose tissue is one of the
sources of mesenchymal stem cells and ADSCs are very similar in nature to bone
mesenchymal stem cells (BMSCs) in terms of multipotency.⁶ Adipose
tissue is abundant in most individuals and can be harvested using a simple
liposuction procedure which is less invasive and cause less discomfort and
donor-site damage. In addition, adipose tissue has a significantly higher stem
cell density than does bone marrow (5% vs 0.01%). ADSCs have been demonstrated
for use in bone, cartilage, and tendon tissue engineering and are used in this
study.

Materials and Methods

The uniaxially-aligned nanofibers were
produced by electrospinning using an air-gap collector based on previous
studies. Poly(ε-caprolactone) (PCL) (Mw=80,000 g/mol; Sigma-Aldrich, St.
Louis, MO) was dissolved in a solvent mixture consisting of dichloromethane
(DCM) and N, N-dimethylformamide (DMF) (Fisher Chemical, Waltham, MA) with a
ratio of 4:1 (v/v) at a concentration of 10% (w/v). Polymer solution was pumped
at a flow rate of 0.5 mL/h using a syringe pump. A dc high voltage of 12 kV was
applied between the nozzle and a grounded air-gap collector made of aluminum
foil (with an open void 2 cm × 5 cm). Subsequently, the uniaxially-aligned
nanofibers were easily transferred to a glass slide. Nanofiber scaffolds were
fabricated using the setup shown in Figure 1. Briefly, a glass slide covered
with uniaxially-aligned nanofibers served as a collector. A movable plastic
mask was connected to a second syringe pump and placed 2 mm above the collector
during deposition of random fibers.

Human ADSCs were obtained from Cellular
Engineering Technologies (Coralville, IA) and were cultured in α-modified
Eagle's medium (α-MEM) supplemented with 10% fetal bovine serum (FBS,
Invitrogen) and 1% gentalmycine/streptomycin (Invitrogen) at 37 °C in an
atmosphere of  95% air / 5% CO₂. Cell culture medium was changed
every 2 days. Prior to cell seeding to nanofiber scaffolds, cells were
trypsinized and counted. Around 1×10⁴ cells were seeded on each
nanofiber scaffold without encapsulation of coumarin 6.

Results and
Discussion

Nanofiber scaffolds with gradations in
fiber organization were generated by deposition of random fibers on an uniaxially-aligned
nanofiber mat. It is observed that more and more random fibers were deposited
from 0 mm to 6 mm along the nanofiber scaffold.  The Fourier fast transfer
(FFT) patterns suggest progression of random fiber generation from 0 to 6 mm. 
In particular the FFT pattern at 6 mm suggests randomly oriented nanofibers.

Once we demonstrated
successful generation of random fiber deposition, we studied whether this gradient
fiber orientation can direct morphological changes following incubation with
ADSCs.  Scaffolds generated using the techniques above were incubated with
ADSCs for 3 and 7 days.  Live cells were detected using a fluorescein diacetate
(FDA) assay (Figure 3, A-H), The response of pre-seeded ADSCs to underlying
nanofiber scaffolds was quantified by measuring the orientation of individual
cells relative to the long axis of the fibers (Figure 3I). The distribution of
cell orientations demonstrated that ADSCs seeded on random nanofibers lacked
organization and directional specificity, as the cells projected in all
possible directions. In contrast, ADSCs seeded on the aligned scaffolds
demonstrated orientation and alignment along the fibers in contact with the
cells. In these cases, approximately 70% of the cells were oriented within 20^o
of the axis of nanofiber alignment. Comparison of the distributions of ADSCs
orientations on the various portions of scaffolds using the Kolmogorov-Smirnov test further confirmed that the pattern of cellular
orientation observed at different portions (distance > 4 mm) on the
nanofiber scaffolds was significantly different  (p < 0.05).

To broaden potential biomedical
applications, chemical gradients were established on this novel scaffold using
nanoencapsulation. Coumarin 6-loaded random PCL nanofibers were generated in
gradient fashion.  We quantified the fluorescence intensity by measuring the
grey scale using Image J software. It is observed that the fluorescent
intensity increased along the distance. This method could be readily extended
to fabricate two-component gradients on the surface by depositing random fibers
twice in two different directions.

Figure 1. Fluorescein
diacetate assay: Adipose-derived stem cells after incubation for 3 days (A-D)
and 7 days (E-H) showing different morphologies on different locations of
nanofiber scaffolds with gradations in fiber organizations (A, E): 0 mm; (B, F): 2 mm; (C, G): 4 mm; and (D, H): 6 mm. (I): Quantitative
analysis of morphology of adipose-derived stem cells on different locations of
nanofiber scaffolds after incubation for 3 days. Compared to the distributions
of cell angle at different locations using the Kolmogorov-Smirnov test, p values of 0.007 (between 0 mm and 6 mm), 0.675 (between 0 mm and 2 mm), 0.031 (between 0 mm and 4 mm), 0.031 (between 2 mm and 6 mm), 0.11 (between 2 mm and 4 mm), and 0.111 (between 4 mm and 6 mm) were obtained. p < 0.05 indicates significant difference.

Conclusion

We have demonstrated a simple, robust
and facile method to fabricate nanofiber scaffolds with gradations in fiber
organization, which could potentially mimic the structure (i.e., collagen fiber
organization) at tendon-to-bone insertion site. We also demonstrated that ADSCs
seeded on such a scaffold exhibited different morphologies at different
locations. In addition, we demonstrated the formation of nanofiber surfaces
with chemical gradients through encapsulation of desired materials inside
deposited nanofibers. By incorporating mineral gradient to this scaffold, it
could be used to recapitulate both structure and composition of tendon-to-bone
insertion site to a considerable degree, which may be promising for the repair
of tendon-to-bone insertion.

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