U.S. patent application number 11/300379 was filed with the patent office on 2006-07-13 for nanofiber construct and method of preparing thereof.
Invention is credited to Kwan-Ho C. Chan, Kazutoshi Fujihara, Masaya Kotaki, Seeram Ramakrishna.
Application Number | 20060154063 11/300379 |
Document ID | / |
Family ID | 38055299 |
Filed Date | 2006-07-13 |
United States Patent
Application |
20060154063 |
Kind Code |
A1 |
Fujihara; Kazutoshi ; et
al. |
July 13, 2006 |
Nanofiber construct and method of preparing thereof
Abstract
The present invention provides a composite nanofiber construct
comprising: at least a first nanofiber comprising at least a
polymer and at least a calcium salt nanoparticle, wherein the ratio
of polymer to calcium salt nanoparticle is between the range of
99:1 and 10:90 weight percent; and at least a second nanofiber
comprising at least a polymer and at least a calcium salt
nanoparticle, wherein the ratio of polymer to calcium salt
nanoparticle is between the range of 100:0 and 70:30 weight
percent. The present invention also provides a method of preparing
the composite nanofiber construct.
Inventors: |
Fujihara; Kazutoshi;
(Singapore, SG) ; Kotaki; Masaya; (Kyoto, JP)
; Ramakrishna; Seeram; (Singapore, SG) ; Chan;
Kwan-Ho C.; (Singapore, SG) |
Correspondence
Address: |
DICKSTEIN SHAPIRO MORIN & OSHINSKY LLP
2101 L Street, NW
Washington
DC
20037
US
|
Family ID: |
38055299 |
Appl. No.: |
11/300379 |
Filed: |
December 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60636356 |
Dec 15, 2004 |
|
|
|
Current U.S.
Class: |
428/373 |
Current CPC
Class: |
A61L 27/18 20130101;
A61L 2400/18 20130101; A61L 2400/12 20130101; A61L 27/18 20130101;
Y10T 428/2929 20150115; D01F 6/625 20130101; D01D 5/0007 20130101;
D01F 1/10 20130101; A61L 27/46 20130101; C08L 67/04 20130101 |
Class at
Publication: |
428/373 |
International
Class: |
D02G 3/00 20060101
D02G003/00 |
Claims
1. A composite nanofiber construct comprising: at least a first
nanofiber comprising at least a polymer and at least a calcium salt
nanoparticle, wherein the ratio of polymer to calcium salt
nanoparticle is between the range of 99:1 and 10:90 weight percent;
and at least a second nanofiber comprising at least a polymer and
at least a calcium salt nanoparticle, wherein the ratio of polymer
to calcium salt nanoparticle is between the range of 100:0 and
70:30 weight percent.
2. The construct according to claim 1, wherein the at least first
nanofiber and the at least second nanofiber have the same or a
different polymer to calcium salt nanoparticle ratio.
3. The construct according to claim 1, wherein the ratio of polymer
to calcium salt nanoparticle of the first nanofiber is 75:25 weight
percent.
4. The construct according to claim 1, wherein the ratio of polymer
to calcium salt nanoparticle of the second nanofiber is 95:5 weight
percent.
5. The construct according to claim 1, wherein the ratio of polymer
to calcium salt nanoparticle of the second nanofiber is 100:0
weight percent.
6. The construct according to claim 1, wherein the average diameter
of the at least first nanofiber is smaller than the average
diameter of the at least second nanofiber.
7. The construct according to claim 1, wherein the at least first
nanofiber and the at least second nanofiber form one structure.
8. The construct according to claim 1, wherein the at least first
nanofiber forms a first structure and the at least second nanofiber
forms a second structure, and wherein the first and second
structures are in contact with each other.
9. The construct according to claim 1, wherein the first nanofiber
and second nanofiber are intertwined.
10. The construct according to claim 7, wherein the structure is a
layer.
11. The construct according to claim 8, wherein the first and
second structures are layers.
12. The construct according to claim 1, wherein the polymer is a
bioabsorbable polymer.
13. The construct according to claim 1, wherein the polymer is
selected from the group consisting of: polycaprolactone,
polyethylene oxide, poly-L-lactic acid, polygyycolide,
poly(DL-lactide), poly(L-lactide), polydioxanone, chitin, collagen
in its native or cross-linked form, poly(glutamic-co-leucine),
poly-lactic-glycolide acid, poly(L-lactic acid-caprolactone)
copolymer and blends, copolymers and terpolymers thereof.
14. The construct according to claim 1, wherein the polymer is
.epsilon.-polycaprolactone.
15. The construct according to claim 1, wherein the calcium salt
nanoparticle is selected from the group consisting of: calcium
carbonate, calcium sulphate, calcium phosphate or
hydroxyapatite.
16. The construct according to claim 15, wherein the calcium salt
is calcium carbonate.
17. The construct according to claim 1, wherein the construct
further comprises seeded cells.
18. The construct according to claim 17, wherein the cells are
selected from the group consisting of osteoblasts, endothelial
cells, smooth muscle cells, mesenchymal stem cells, embryonic stem
cells, chondroblasts, fibrocytes, fibroblasts and chondrocytes.
19. The construct according to claim 1, wherein the construct is an
implant.
20. The construct according to claim 1, wherein the construct is
surface functionalized.
21. The construct according to claim 20, wherein the construct is
surface functionalized by plasma treatment.
22. A method of preparing a composite nanofiber construct, the
method comprising the steps of: preparing at least a first
nanofiber comprising at least a polymer and at least a calcium salt
nanoparticle, wherein the ratio of polymer to calcium salt
nanoparticles is between the range of 99:1 and 10:90 weight
percent; preparing at least a second nanofiber comprising at least
a polymer and at least a calcium salt nanoparticle, wherein the
ratio of polymer to calcium salt nanoparticle is between the range
of 100:0 and 70:30 weight percent; and preparing a composite
nanofiber construct by contacting the first nanofiber with the
second nanofiber.
23. The method according to claim 22, wherein the at least first
nanofiber and the at least second nanofiber have the same or a
different polymer to calcium salt nanoparticle ratio.
24. The method according to claim 22, wherein the first nanofiber
is prepared by adding calcium salt to a solvent and mixing the
resulting mixture with the first polymer obtaining a first solvent
mixture; and the second nanofiber is prepared by adding calcium
salt to a solvent and mixing the resulted mixture with the second
polymer obtaining a second solvent mixture.
25. The method according to claim 22, wherein the first nanofiber
is prepared by adding calcium salt to a solvent and mixing the
resulting mixture with the first polymer obtaining a first solvent
mixture; and the second nanofiber is prepared by adding the second
polymer to a solvent obtaining a second solvent mixture.
26. The method according to claim 24, wherein the first and second
solvent mixtures comprise methanol and/or chloroform.
27. The method according to claim 25, wherein the first and second
solvent mixtures comprise methanol and/or chloroform.
28. The method according to claim 22, wherein the ratio of polymer
to calcium salt nanoparticle of the second nanofiber is 100:0
weight percent.
29. The method according to claim 22, wherein the average diameter
of the at least first nanofiber is smaller than the average
diameter of the at least second nanofiber.
30. The method according to claim 24, wherein the first solvent
mixture and second solvent mixture are provided simultaneously, and
wherein the at least first nanofiber and the at least second
nanofiber are intertwined.
31. The method according to claim 25, wherein the first solvent
mixture and the second solvent mixture are provided consecutively,
and wherein the at least first nanofiber and the at least second
nanofiber are separate from each other.
32. The method according to claim 22, wherein the method further
comprises the step of surface functionalizing the construct.
33. A kit comprising a composite nanofiber construct comprising: at
least a first nanofiber comprising at least a polymer and at least
a calcium salt nanoparticle, wherein the ratio of polymer to
calcium salt nanoparticle is between the range of 99:1 and 10:90
weight percent; and at least a second nanofiber comprising at least
a polymer and at least a calcium salt nanoparticle, wherein the
ratio of polymer to calcium salt nanoparticle is between the range
of 100:0 and 70:30 weight percent.
34. A method of repairing fractured bone segments in a subject,
comprising the step of surgically implanting the construct of claim
1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/636,356, filed on Dec. 15, 2004, the entirety of
the contents of which are hereby incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] The present invention relates to a nanofiber construct and
method of preparing the same.
BACKGROUND OF THE INVENTION
[0003] Different types of polymer fibers with nanometer scale
diameter have been recently prepared by electrospinning method. As
compared to the conventional polymer fibers with micrometer scale,
nanofibers have a high surface area-to-volume ratio. Hence,
electrospun nanofibers appear to have better potential in several
bioengineering applications, such as tissue regeneration,
biosensors, recognition and filtration of viruses and drug
molecules.
[0004] The interaction between nanofiber scaffolds and
proliferation cells, like human osteoblasts, smooth muscle cells,
mesenchymal stem cells and chondrocytes have been investigated and
the feasibility of nanometer scale polymer fibers as tissue
scaffolds have been studied. However, nanometer scale dimension is
not the only factor which encourages cell attachment and growth. It
is known that human osteoblasts cannot attach to hydrophobic
surfaces whereas endothelial cells can attach to hydrophobic
surfaces.
[0005] Fujihara et al., Eight Japan International SAMPE Symposium
and Exhibition, 2003, p. 1213-6, disclosed the preparation of
guided bone regeneration (GBR) membranes fabricated by
polycaprolactone and calcium carbonate nanofibers with PLC to
calcium carbonate 90:10 wt % ratio. They observed that calcium rich
membranes were preferred in vivo conditions as they enhance
osteoconductivity of bone defects. However, they also found that
the tensile property of these membranes decreased with the increase
of amounts of calcium carbonate particles. Accordingly, these
membranes were not suitable for implant uses as they are
mechanically not stable.
[0006] Accordingly, although prior art teaches electrospinning of
polymer nanofibers, there exists a need to fabricate stable polymer
composite nanofibers suitable as scaffolds for tissue engineering.
In particular, there is a need to create improved composite
nanofibrous membranes with appropriate composition and surface
modification for application in tissue engineering in which cell
attachment and growth are enhanced.
SUMMARY OF THE INVENTION
[0007] The present invention addresses the problems above, and in
particular, provides a new composite nanofiber construct and method
of producing the said construct.
[0008] According to a first aspect, the present invention provides
a composite nanofiber construct comprising: [0009] at least a first
nanofiber comprising at least a polymer and at least a calcium salt
nanoparticle, wherein the ratio of polymer to calcium salt
nanoparticle may be between the range of 99:1 and 10:90 weight
percent; and [0010] at least a second nanofiber comprising at least
a polymer and at least a calcium salt nanoparticle, wherein the
ratio of polymer to calcium salt nanoparticle may be between the
range of 100:0 and 70:30 weight percent.
[0011] The at least first nanofiber and the at least second
nanofiber may have the same polymer to calcium salt nanoparticle
ratio, or the at least first nanofiber and the at least second
nanofiber may have a different polymer to calcium salt nanoparticle
ratio. According to a particular aspect, the at least second
nanofiber has a different polymer to calcium salt nanoparticle
ratio from the at least first nanofiber. In particular, the at
least second nanofiber has a higher polymer to calcium salt
nanoparticle ratio than the at least first nanofiber. For example,
the polymer to calcium salt nanoparticle ratio of the at least
first nanofiber may be 75:25 and the polymer to calcium salt
nanoparticle ratio of the at least second nanofiber may be 95:5,
99:1 or 100:0. According to another particular aspect, the at least
first nanofiber and the at least second nanofiber have the same
polymer to calcium salt nanoparticle ratio. For example, the
polymer to calcium salt nanoparticle ratio may be 75:25 weight
percent.
[0012] The first nanofiber is also known as functionalised
nanofiber. The second nanofiber is known as support nanofiber. The
support nanofiber may comprise calcium salt. According to one
aspect, the support nanofiber only comprises small amounts or
traces of calcium salt. According to another aspect, the support
nanofiber does not comprise calcium salt.
[0013] According to another aspect, the average diameter of the at
least first nanofiber may be the same as or different from the
average diameter of the at least second nanofiber. In particular,
the average diameter of the at least first nanofiber is smaller
than the average diameter of the at least second nanofiber.
[0014] The construct of the invention may be a single structure
(for example, a layer) formed by the at least first nanofiber and
the at least second nanofiber. Alternatively, the at least first
nanofiber may form a first structure (for example, a first layer)
and the at least second nanofiber may form a second structure (for
example, a second layer), and wherein the first and second
structures may be in contact with each other to form the construct.
In particular, the first nanofiber and second nanofiber may be
intertwined.
[0015] Accordingly, the structure may be a layer, or the structure
may be such that the first and second structures are layers.
According to one aspect, the construct according to the invention
comprises at least a layer comprising functionalised (first)
nanofiber(s) supported by at least a support layer comprising
support (second) nanofiber(s). According to another aspect, the
functionalised nanofiber(s) and support nanofiber(s) are combined
to form a single layer. For example, the functionalised
nanofiber(s) and support nanofiber(s) are deposited simultaneously
to form an intertwined layer. The construct according to the
invention may comprise more than one of said layer.
[0016] The nanofiber(s), solvent mixture(s), construct(s), implant,
kit and method according to the invention comprises the use of at
least one polymer. Accordingly, a mixture of polymers may be used.
The polymer may be a bioabsorbable polymer. The polymer may be
selected from the group consisting of: polycaprolactone,
polyethylene oxide, poly-L-lactic acid, polygyycolide,
poly(DL-lactide), poly(L-lactide), polydioxanone, chitin, collagen
either in its native form or cross-linked,
poly(glutamic-co-leucine), poly-lactic-glycolide acid,
poly(L-lactic acid-caprolactone) copolymer and blends, copolymers
and terpolymers thereof. In particular, the polymer is
.epsilon.-polycaprolactone.
[0017] The nanofiber(s), solvent mixture(s), construct(s), implant,
kit and method according to the invention comprises the use of at
least one calcium salt. Accordingly, more than one calcium salt or
a mixture of calcium salts may be used in the present invention.
The calcium salt may be selected from the group consisting of:
calcium carbonate, calcium sulphate, calcium phosphate or
hydroxyapatite. In particular, the calcium salt is calcium
carbonate. However, more than one type of calcium salt or a mixture
thereof may be used.
[0018] The construct may further comprise seeded cells. The cells
may be selected from the group consisting of osteoblasts,
endothelial cells, smooth muscle cells, mesenchymal stem cells,
embryonic stem cells, chondroblasts, fibrocytes, fibroblasts and
chondrocytes. In particular, the cells are human osteoblasts.
[0019] According to a further aspect, the construct is an implant
or a scaffold.
[0020] The present invention also relates to the functionalization
of nanofibers to make it suitable for use as tissue scaffolds and
other applications in tissue engineering. Therefore, according to
another further aspect, the construct may be surface
functionalized. For example, the construct may be surface
functionalized by polymer grafting and/or plasma treatment. The
construct may also be surface functionalized by dipping and washing
the construct in sodium hydroxide solution. In particular, the
construct is surface functionalized by plasma treatment.
[0021] Accordingly, the present invention also provides the
fabrication method and/or surface modification of composite
nanofiber constructs. The invention enhances cell attachment and
proliferation and by creating a suitable construct the mechanical
strength of the construct can be appropriately tailored to meet the
intended use.
[0022] The present invention, in particular, discloses a method of
fabricating nanofibers suitable for use as scaffold for
osteoblasts. This invention discloses methods of fabrication and/or
surface modification of biodegradable and/or bioabsorbable polymer
composite nanofibers. In particular, the construct according to the
invention may have at least the following characteristics: (a) the
composition of composite nanofibers can be tailored to the
proliferation and attachment of different cell types; and (b) the
mechanical properties and biochemical properties can be adjusted
independently by electrospinning, surface functionalization and the
addition of filler particles or nanoparticles such as calcium salt
nanoparticles.
[0023] Accordingly, there is also provided a method of preparing
the composite nanofiber construct according to any aspect of the
invention, the method comprising the steps of: [0024] preparing at
least a first nanofiber comprising at least a polymer and at least
a calcium salt nanoparticle, wherein the ratio of polymer to
calcium salt nanoparticle is between the range of 99:1 and 10:90
weight percent; [0025] preparing at least a second nanofiber
comprising at least a polymer and at least a calcium salt
nanoparticle, wherein the ratio of polymer to calcium salt
nanoparticle is between the range of 100:0 and 70:30 weight
percent; and [0026] preparing a composite nanofiber construct by
contacting the first nanofiber with the second nanofiber.
[0027] The first nanofiber may be prepared by adding at least a
calcium salt to at least a solvent and mixing the resulting mixture
with the at least first polymer obtaining a first solvent mixture;
and the second nanofiber may be prepared by adding at least a
calcium salt to at least a solvent and mixing the resulted mixture
with the at least second polymer obtaining a second solvent
mixture. The second nanofiber may also be prepared by adding the
second polymer to a solvent to obtain a second solvent mixture. In
the latter case, no calcium salt is added to the mixture.
[0028] Accordingly, the method of the invention may comprise the
step of preparing a first solvent mixture and a second solvent
mixture. The solvent of first and second solvent mixtures may be
methanol and/or chloroform. Other suitable solvents may also be
used.
[0029] According to a particular aspect, the at least second
nanofiber has a different polymer to calcium salt nanoparticle
ratio from the at least first nanofiber. In particular, the at
least second nanofiber has a higher polymer to calcium salt
nanoparticle ratio than the at least first nanofiber. For example,
the polymer to calcium salt nanoparticle ratio of the at least
first nanofiber may be 75:25 and the polymer to calcium salt
nanoparticle ratio of the at least second nanofiber may be 100:0.
According to another particular aspect, the at least first
nanofiber and the at least second nanofiber have the same polymer
to calcium salt nanoparticle ratio. For example, the polymer to
calcium salt nanoparticle ratio may be 75:25 weight percent. In
particular, the ratio of polymer to calcium salt nanoparticle of
the first nanofiber is 75:25 weight percent. In particular, the
ratio of polymer to calcium salt nanoparticle of the second
nanofiber is 95:5 weight percent. Even more in particular, the
ratio of polymer to calcium salt nanoparticle of the second
nanofiber is 100:0 weight percent.
[0030] According to another aspect, the first solvent mixture may
be provided to form the at least first nanofiber and the second
solvent mixture may be provided to form the at least second
nanofiber.
[0031] According to another aspect, the average diameter of the at
least first nanofiber may be the same as or different from the
average diameter of the at least second nanofiber. In particular,
the average diameter of the at least first nanofiber is smaller
than the average diameter of the at least second nanofiber.
[0032] The first solvent mixture and second solvent mixture may be
provided simultaneously such that the at least first nanofiber and
the at least second nanofiber are intertwined. Alternatively, the
first solvent mixture and second solvent mixture may be provided
sequentially one after another such that the at least first
nanofiber and the at least second nanofiber are separate from each
other. In particular, the first solvent mixture and second solvent
mixture may be provided to form the at least first nanofiber and
the at least second nanofiber respectively.
[0033] The method of the invention may further comprise the step of
seeding the construct with cells. The cells may be selected from
the group consisting of osteoblasts, endothelial cells, smooth
muscle cells, mesenchymal stem cells, embryonic stem cells,
chondroblasts, fibrocytes, fibroblasts and chondrocytes. In
particular, the cells are human osteoblasts.
[0034] The method of the invention may further comprise the step of
surface functionalizing the construct. The step of surface
functionalising may comprise polymer grafting and/or plasma
treatment. The step of surface functionalizing may also comprise
dipping and washing the construct in sodium hydroxide solution. In
particular, the step of surface functionalising comprises plasma
treatment of the construct. Other known methods of surface
functionalization suitable for the purposes of the present
invention are also encompassed by the construct and/or method of
the present invention.
[0035] Therefore, a fabrication method and/or surface modification
of polymer composite nanofiber construct(s) is provided. The method
comprises the preparation of polymer solution comprising filler
particles such as calcium salt particles, the principle of
electrospinning method and preferably air-plasma treatment to
enhance the hydrophilicity of composite nanofiber constructs.
Additionally, nanofibers constructs are formed by electrospinning
two or more types of nanofibers either simultaneously or
sequentially in layers. In the composite nanofiber construct, the
diameter and composition of one type of nanofiber may be adjusted
for cell attachment and growth while the diameter and composition
of the other may be adjusted for mechanical strength. In another
type of composite nanofiber construct, the diameter and composition
of each type of nanofiber is selected for cell attachment and
proliferation for specific desired cell types. Other aspects,
features and advantages of the invention will become apparent to
those of ordinary skill in the art upon review of the description
of specific embodiments of the invention. Calcium salt
nanoparticles are added to the solvent mixture to result in
composite nanofibers to enhance cell attachment, in particular
osteoblast attachment. Such composite nanofibers are suitable for
use as bone graft substitutes. The addition of calcium salt
nanoparticles enhances the osteoconductive property of the
nanofibers. Furthermore, human osteoblasts prefer a calcium rich
environment. Composite nanofibers embedded with calcium phosphate
can enhance cell attachment and growth. Besides nanometer scale
fiber constructs, functionalization (surface modification) of
nanofibers is of paramount importance to promote better cell fiber
interaction.
[0036] There is also provided a kit comprising the construct of the
present invention. In particular, the kit comprises: [0037] at
least a first nanofiber comprising at least a polymer and at least
a calcium salt nanoparticle, wherein the ratio of polymer to
calcium salt nanoparticle is between the range of 99:1 and 10:90
weight percent; and [0038] at least a second nanofiber comprising
at least a polymer and at least a calcium salt nanoparticle,
wherein the ratio of polymer to calcium salt nanoparticle is
between the range of 100:0 and 70:30 weight percent.
[0039] The kit according to the invention may further comprise
instructions for use(s) and/or application(s) of the construct,
scaffold and/or implant according to the invention.
[0040] The present invention further provides a method of repairing
fractured bone segment(s) in a subject, comprising the step of
surgically implanting the construct of the present invention.
[0041] The present invention also provides a method of fabricating
composite nanofibers constructs for use as tissue engineering
scaffold and/or bone void fillers comprising the following steps:
[0042] a) dispersing particles of calcium salt in a solvent; [0043]
b) adding a polymer to the resultant mixture from (a) and agitating
until the polymer is completely dissolved; [0044] c) the resultant
polymer solution containing calcium salt particles of (b) is
dispensed through one or more outlets from one or more dispensers
at a predetermined rate under controlled humidity and at a
predetermined height separating the said discharge outlets from a
collector plate; [0045] d) a voltage is applied between the lowest
point of the dispenser and a collector plate; whereby application
of voltage draws the resultant mixture (c) into fine elongations
and with simultaneous evaporation of the solvent resulting in the
deposition of composite nanofibers on the collector plate.
[0046] The method further includes drying of the said composite
nanofiber constructs sufficient for all the solvent to evaporate.
The solvent is a mixture of chloroform and methanol. Further, the
polymer is a bioabsorbable polymer. The polymer may be selected
from lactone, polyethylene oxide, poly-L-lactic acid,
poly-lactic-glycolide acid and poly (L-lactic acid-caprolactone)
copolymer.
[0047] Further, the said calcium salt is any suitable combination
of one or more calcium salt selected from the following: calcium
carbonate, calcium sulphate, calcium phosphate or hydroxyapatite.
The nanoparticles of calcium salt is between 5 nm and 1000 nm in
its largest dimension.
[0048] The said solvent may vary in composition from 95 weight
percent chloroform and 5 weight percent methanol to 5 weight
percent chloroform and 95 weight percent methanol. The mass of
calcium salt nanoparticle may vary from 1 g to 50 g per 100 g of
the said solvent. The mass of polycaprolactone may vary from 1
weight percent to 40 weight percent.
[0049] The dispenser in step (c) is any container with one or more
outlets and wherein the outlet diameter ranges from 0.05 to 0.9 mm.
In particular, the dispenser is a syringe connected to a hypodermic
needle. The hypodermic needle may be a #27 gauge. The method may
further comprise a dispenser controller for controlling the
dispensing of the resultant mixture of step (b). The dispensing
rate may be between 0.1 ml to 10 ml per outlet per hour.
[0050] The predetermined height separating the discharge outlets
from the collector plate may vary from 1 mm to 1 meter. There may
be multiple dispensers connecting to one or more outlets. Further,
each dispenser may dispense different formulations of the mixture
from step (b). In particular, one or more dispenser dispenses
bioabsorbable polymer without calcium salt.
[0051] The method may further include the step of air plasma
treatment of the composite nanofibers. The method comprises the
step of storing the composite nanofibers in aqueous solution until
it is ready for use.
BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1 (a) shows an electrospinning apparatus of polymer
nanofibers with one dispenser and outlet.
[0053] FIG. 1 (b) shows an electrospinning apparatus of polymer
nanofibers with two dispensers and outlets.
[0054] FIG. 2 shows a two-layer structure of composite nanofiber
construct.
[0055] FIG. 3 shows air-plasma treatment of composite nanofiber
constructs.
[0056] FIG. 4 shows SEM (Scanning Electron Microscope) photographs
of (a) composite nanofiber construct comprising PCL and (b)
composite composite nanofibers comprising PCL/CaCO.sub.3.
[0057] FIG. 5 shows EDX (Energy Disperse X-ray) mapping of (a)
composite nanofiber construct comprising PCL and (b) composite
nanofibers comprising PCL/CaCO.sub.3 (PCL:CaCO.sub.3=25:75).
[0058] FIG. 6 shows osteoblast attachment manner on composite
nanofiber scaffolds.
[0059] FIG. 7 shows SEM photographs of PCL nanofibers made of (A)
PCL 5 weight percent solution and (B) PCL 7.5 weight percent
solution.
[0060] FIG. 8 shows SEM photographs of PCL/CaCO.sub.3 nanofibers
(PCL:PCL/CaCO.sub.3=25:75) made of (A) PCL 3 weight percent
solution and (B) PCL 5 weight percent solution.
[0061] FIG. 9 shows the absorbance intensity at 490 nm of TCPS and
the construct against seeding time.
[0062] FIG. 10 shows osteoblast attachment manner on the
construct.
[0063] FIG. 11 shows a two-layer structure of the construct to
avoid rupture during cell seeding procedure. Two different
composite nanofibers (i.e. GBR membrane (A) PCL:CaCO.sub.3=75:25
and GBR membrane (B) PCL:CaCO.sub.3=25:75) were prepared for
osteoblast seeding.
[0064] FIG. 12 shows the tensile stress-strain curve of nanofiber
(B). Visible rupture was not recognized at 200% apparent
strain.
DETAILED DESCRIPTION OF THE INVENTION
[0065] The whole contents of any bibliographic reference is herein
incorporated by reference.
[0066] According to a first aspect, the present invention provides
a composite nanofiber construct comprising: [0067] at least a first
nanofiber comprising at least a polymer and calcium salt
nanoparticle, wherein the ratio of polymer to calcium salt
nanoparticle is between the range of 99:1 and 10:90 weight percent;
and [0068] at least a second nanofiber comprising at least a
polymer and calcium salt nanoparticle, wherein the ratio of polymer
to calcium salt nanoparticle is between the range of 100:0 and
70:30 weight percent.
[0069] It will be evident to a skilled person that the at least
first nanofiber and the at least second nanofiber are
distinguishable from each other. For example, the at least first
nanofiber and the at least second nanofiber may have different
physical and/or chemical characteristics. The physical and/or
chemical characteristic may include, but is not limited to, polymer
to calcium salt nanoparticle ratio, diameter, surface
functionalization treatment and treatment to adapt the nanofiber to
a particular function.
[0070] Nanofibers may comprise fibers ranging in diameter from
approximately 1 nanometer (nm) (10.sup.-9 meters) to approximately
10000 nm. In particular, the fibers range in diameter from 10 nm to
10000 nm, preferably from 200 nm to 1500 nm.
[0071] According to a particular aspect, the average diameter of
the section(s) of the at least first nanofiber may be the same as
or different from the average diameter of the section(s) of the at
least second nanofiber. In particular, the average diameter of the
at least first nanofiber is smaller than the average diameter of
the at least second nanofiber. The at least first nanofiber
preferably has an average diameter between 10 and 1000 nm, more
preferably between 20 and 500 nm, and even more preferably, 25 to
100 nm. In particular, the average diameter is about 50 nm, more in
particular, 50 nm. The at least second nanofiber preferably has an
average diameter between 10 and 1000 nm, more preferably between 20
and 500 nm, and even more preferably, 100 to 400 nm. In particular,
the average diameter is about 300 nm, more in particular 300
nm.
[0072] The ratio of polymer to calcium salt nanoparticle of the at
least first nanofiber may be between 99:1 to 10:90, 80:20 to 20:80,
75:25 to 25:75, 70:30 to 30:70, 60:40 and 40:60 or 50:50. In
particular, the ratio of polymer to calcium salt nanoparticle of
the first nanofiber is 70:30 weight percent. More in particular,
the ratio of polymer to calcium salt nanoparticle of the first
nanofiber is 75:25.
[0073] The ratio of polymer to calcium salt nanoparticle of the at
least second nanofiber may be between 100:0 to 70:30, 95:5 to
80:20, 90:10 to 85:15 or 85:15 to 80:20. In particular, the ratio
of polymer to calcium salt nanoparticle of the second nanofiber is
95:5 weight percent. More in particular, the ratio of polymer to
calcium salt nanoparticle of the second nanofiber is 100:0 weight
percent.
[0074] According to a particular aspect, the at least second
nanofiber has a different polymer to calcium salt nanoparticle
ratio from the at least first nanofiber. In particular, the at
least second nanofiber has a higher polymer to calcium salt
nanoparticle ratio than the at least first nanofiber. For example,
the polymer to calcium salt nanoparticle ratio of the at least
first nanofiber may be 75:25 weight percent and the polymer to
calcium salt nanoparticle ratio of the at least second nanofiber
may be 100:0 weight percent. According to another particular
aspect, the at least first nanofiber and the at least second
nanofiber have the same polymer to calcium salt nanoparticle ratio.
For example, the polymer to calcium salt nanoparticle ratio may be
75:25 weight percent.
[0075] In particular, the at least first nanofiber and the at least
second nanofiber have the same polymer to calcium salt nanoparticle
ratio, but the average diameter of the at least first nanofiber and
the at least second nanofiber are different. For example, the ratio
of polymer to calcium salt nanoparticle in the at least first and
second naofibers is 75:25, and the average diameter of the at least
first nanofiber is approximately 50 nm while the average diameter
of the at least second nanofiber is approximately 300 nm. The at
least second nanofiber provides mechanical support to the construct
by virtue of its larger diameter.
[0076] The at least first nanofiber and the at least second
nanofiber may form one structure or the at least first nanofiber
may form a first structure and the at least second nanofiber may
form a second structure, and wherein the first and second
structures may be in contact with each other. In particular, the
first nanofiber and second nanofiber may be intertwined.
[0077] The structure may be a layer, or the structure may be such
that the first and second structures are separate layers. According
to one aspect, the construct according to the invention comprises
at least a layer comprising functionalised (first) nanofiber(s)
supported by at least a support layer comprising support (second)
nanofiber(s). According to another aspect, the functionalised
nanobifer(s) and support nanofiber(s) are combined to form a single
layer. For example, the functionalised nanofiber(s) and support
nanofiber(s) are deposited simultaneously to form an intertwined
layer. The construct according to the invention may comprise more
than one of said layer.
[0078] The polymer may be a bioabsorbable polymer. The polymer may
be selected from the group consisting of: polycaprolactone,
polyethylene oxide, poly-L-lactic acid, polygyycolide,
poly(DL-lactide), poly(L-lactide), polydioxanone, chitin, collagen
either in its native form or cross-linked,
poly(glutamic-co-leucine), poly-lactic-glycolide acid,
poly(L-lactic acid-caprolactone) copolymer and blends, copolymers
and terpolymers thereof. In particular, the polymer is
.epsilon.-polycaprolactone. However, any suitable polymer may be
used. Further, the at least first nanofiber and the at least second
nanofiber may comprise one type of polymer or a mixture of two or
more polymers.
[0079] The present invention also provides that the calcium salt
nanoparticle is selected from the group consisting of: calcium
carbonate, calcium sulphate, calcium phosphate or hydroxyapatite.
In particular, the calcium salt is calcium carbonate. However, any
suitable calcium salt may be used. Further, the at least first
nanofiber and the at least second nanofiber may comprise one type
of calcium salt or a mixture of two or more calcium salts.
[0080] The construct may further comprise seeded cells. The cells
may be selected from the group consisting of osteoblasts,
endothelial cells, smooth muscle cells, mesenchymal stem cells,
embryonic stem cells, chondroblasts, fibrocytes, fibroblasts and
chondrocytes. In particular, the cells are human osteoblasts.
However, any suitable cell may be used. Further, the construct may
comprise more than one type of cell.
[0081] According to a further aspect, the construct is an implant
or a scaffold. The construct may be used in various applications
including, but not limited to, tissue engineering, bone graft
substitute and/or periodontal regeneration. The construct may also
be used for non-therapeutic and/or cosmetic purposes. It would be
known to a skilled person how to use the construct. For example,
the construct may be used in, but not limited to, non-therapeutic
and/or cosmetic periodontal purposes. It is important for an
implant to be capable of both osteointegration and osteoconduction.
Osteointegration refers to direct chemical bonding of a biomaterial
to the surface of bone without an intervening layer of fibrous
tissue. This bonding is referred to as the implant-bone interface.
A primary problem with skeletal implants is mobility.
Osteoconduction refers to the ability of a biomaterial to sustain
cell growth and proliferation over its surface while maintaining
the cellular phenotype. For osteoblasts, the phenotype includes
mineralization, collagen production, and protein synthesis. Normal
osteoblast function is particularly important for porous implants
that require bone ingrowth for proper strength and adequate surface
area for bone bonding. In addition, implants may be both
biocompatible and biodegradable.
[0082] According to a further aspect, the present invention also
relates to the functionalization of nanofibers to make it suitable
for use as tissue scaffolds and other applications in tissue
engineering. Therefore, the present invention provides a surface
functionalised scaffold. For example, the construct may be surface
functionalized by polymer grafting and/or plasma treatment. The
construct may also be surface functionalized by dipping and washing
the construct in sodium hydroxide solution. In particular, the
construct is surface functionalized by plasma treatment.
[0083] Hydrophobicity of material is also necessary for cell
attachment and proliferation. Plasma treatment is a useful method
to modify the surface-chemical structure of polymers and its most
apparent effect is modified wettability. According to Liston et al,
Plasma surface modification of polymers for improved adhesion: a
critical review, 1994, VSP BV Netherlands, plasma-produced polar
chemical groups increase the surface energy of polymer and decrease
in surface contact angle. Lee et al, Cell adhesion and growth on
polymer surfaces with hydroxyl groups prepared by water vapour
plasma treatment, Biomaterials, 1991, 12:443-8, investigated the
interaction between plasma-modified polymers and ovary cell
behaviour. It was shown that hydroxyl groups are mainly produced on
the surface of polymers and this surface modification resulted in
the decrease of surface contact angle which led to good adhesion
and growing manners of cells.
[0084] Contact angle analysis characterises the wettability of a
surface by measuring the surface tension of a solvent droplet at
its interface with a homogenous surface. In more technical terms,
contact angle measures the attraction or repulsion those droplet
molecules experience towards the surface molecules. Any suitable
method may be used for measuring the contact angle.
[0085] Further, the present invention also provides a method of
preparing the composite nanofiber construct of the present
invention, the method comprising the steps of: [0086] preparing at
least a first nanofiber comprising at least a polymer and at least
a calcium salt nanoparticle, wherein the ratio of polymer to
calcium salt nanoparticle is between the range of 99:1 and 10:90
weight percent; [0087] preparing at least a second nanofiber
comprising at least a polymer and at least a calcium salt
nanoparticle, wherein the ratio of polymer to calcium salt
nanoparticle is between the range of 100:0 and 70:30 weight
percent; and [0088] preparing a composite nanofiber construct by
contacting the first nanofiber with the second nanofiber.
[0089] The method according to the invention may further comprise
the step of preparing a first solvent mixture and a second solvent
mixture. The first solvent mixture may be used to prepare the at
least first nanofiber and the second solvent mixture may be used to
prepare the at least second nanofiber. The first and second solvent
mixtures may comprise methanol and/or chloroform. However, the
composition of the solvent mixtures may vary to suit the needs of
the present invention. For example, any one of the solvents, or a
combination thereof, as listed in Zheng-Ming Huang et al, A review
on polymer nanofibers by electrospinning and their applications in
nanocomposites, Composites Science and Technology, 2003,
63:2223-2253, may be used.
[0090] According to a further aspect, the ratio of polymer to
calcium salt nanoparticle of the first nanofiber may be between
99:1 to 10:90, 80:20 to 20:80, 75:25 to 25:75, 70:30 to 30:70,
60:40 and 40:60 or 50:50. In particular, the ratio of polymer to
calcium salt nanoparticle of the first nanofiber is 70:30 weight
percent. Even more in particular, the ratio of polymer to calcium
salt nanoparticle of the first nanofiber is 75:25.
[0091] According to a further aspect, the ratio of polymer to
calcium salt nanoparticle of the second nanofiber may be between
100:0 to 70:30, 95:5 to 80:20, 90:10 to 85:15 or 85:15 to 80:20. In
particular, the ratio of polymer to calcium salt nanoparticle of
the second nanofiber is 95:5 weight percent. Even more in
particular, the ratio of polymer to calcium salt nanoparticle of
the second nanofiber is 100:0 weight percent.
[0092] According to a particular aspect, the at least second
nanofiber has a different polymer to calcium salt nanoparticle
ratio from the at least first nanofiber. In particular, the at
least second nanofiber has a higher polymer to calcium salt
nanoparticle ratio than the at least first nanofiber. For example,
the polymer to calcium salt nanoparticle ratio of the at least
first nanofiber may be 75:25 weight percent and the polymer to
calcium salt nanoparticle ratio of the at least second nanofiber
may be 100:0 weight percent. According to another particular
aspect, the at least first nanofiber and the at least second
nanofiber have the same polymer to calcium salt nanoparticle ratio.
For example, the polymer to calcium salt nanoparticle ratio may be
75:25 weight percent.
[0093] According to another aspect, the average diameter of the at
least first nanofiber may be the same as or different from the
average diameter of the at least second nanofiber. In particular,
the average diameter of the at least first nanofiber is smaller
than the average diameter of the at least second nanofiber.
[0094] The at least first nanofiber preferably has an average
diameter between 10 and 1000 nm, more preferably between 20 and 500
nm, and even more preferably, 25 to 100 nm. In particular, the
average diameter is about 50 nm, more in particular 50 nm. The at
least second nanofiber preferably has an average diameter between
10 and 1000 nm, more preferably between 20 and 500 nm, and even
more preferably, 100 to 400 nm. In particular, the average diameter
is about 300 nm, more in particular 300 nm.
[0095] In particular, the at least first nanofiber and the at least
second nanofiber have the same polymer to calcium salt nanoparticle
ratio, but the average diameter of the at least first nanofiber and
the at least second nanofiber are different. For example, the ratio
of polymer to calcium salt nanoparticle in the at least first and
second naofibers is 75:25, and the average diameter of the at least
first nanofiber is approximately 50 nm while the average diameter
of the at least second nanofiber is approximately 300 nm.
[0096] The polymer may be a bioabsorbable polymer. The polymer may
be selected from the group consisting of: polycaprolactone,
polyethylene oxide, poly-L-lactic acid, polygyycolide,
poly(DL-lactide), poly(L-lactide), polydioxanone, chitin, collagen
either in its native form or cross-linked,
poly(glutamic-co-leucine), poly-lactic-glycolide acid,
poly(L-lactic acid-caprolactone) copolymer and blends, copolymers
and terpolymers thereof. In particular, the polymer is
.epsilon.-polycaprolactone. However, any suitable polymer may be
used. Further, the at least first nanofiber and the at least second
nanofiber may comprise one type of polymer or a mixture of two or
more polymers.
[0097] The present invention also provides that the at least
calcium salt nanoparticle is selected from the group consisting of:
calcium carbonate, calcium sulphate, calcium phosphate or
hydroxyapatite. In particular, the calcium salt is calcium
carbonate. However, any suitable calcium salt may be used. Further,
the at least first nanofiber and the at least second nanofiber may
comprise one type of calcium salt or a mixture of two or more
calcium salts.
[0098] According to a particular aspect, the method of the present
invention comprises electrospinning the at least first nanofiber
and the at least second nanofiber. Electrospinning apparatus to
make nanofibers generally consists of a high voltage power supply,
a dispenser pump with feeding rate controller, a dispenser in the
form of a syringe containing a solvent mixture, an outlet in the
form of a needle with small diameter hole and a fiber collecting
plate, as shown in FIG. 1A. In the electrospinning process, a high
voltage is used to create an electrically charged jet of polymer
solution. Generally, a positive charge is applied to the solvent
mixture at the outlet end while the fiber collector plate is
grounded. Because of the high voltage, an intense electrical field
is generated between the outlet and fiber collector plate. When the
electrical force exceeds the surface tension of the solvent
mixture, jets of solvent mixture are drawn towards to the fiber
collecting plate. The solvent mixture jet is stretched to nanometer
scale by electrical force and the solvent evaporates from the
stream of solvent mixture jets to form solid nanofibers. Polymer
nanofiber membranes are formed by the deposition of the nanofibers
on the collector plate.
[0099] FIG. 1A shows a dispenser filled with a solvent mixture. The
feed rate of the dispenser is controlled by a dispenser pump. A
flexible plastic tube connects the dispenser to an outlet, which
can be in the form of a small orifice such as a small bore
hypodermic needle. The outlet is usually clamped to a stand. Using
a high-voltage power supply, voltage is applied to the outlet tip
at room temperature with controlled humidity conditions. For
example, the humidity is set to 30 to 40% to control the
evaporation rate of the solvent. The electrically charged solvent
mixture forms a Taylor cone from the tip of the outlet to the
grounded collector plate at a fixed distance. The fine jet of
electrically charged solvent mixture is drawn towards the grounded
collector plate. The elongation of the solvent mixture jet results
in decreasing diameter of the jet and travels towards the collector
plate in a spiral fashion. During this travel, the solvent in the
solvent mixture evaporates in the air and randomly oriented
nanofibers are deposited on the collector plate. Generally, the
mechanical properties of composite nanofibers are weaker than that
of nanofibers which comprise added particles, such as calcium salt
nanoparticles.
[0100] Depending on the composition of the calcium salt
nanoparticles in the solvent mixtures, the formed nanofiber may be
fragile and difficult to handle, particularly when a large amount
of calcium salt nanoparticles are added. To enhance the handling
characteristic of the nanofiber construct, a composite nanofiber
structure may be fabricated by a number of methods as described
below.
[0101] According to a further aspect, the first solvent mixture is
provided to form the at least first nanofiber and the second
solvent mixture is provided to form the at least second nanofiber.
The first solvent mixture and second solvent mixture may be
provided simultaneously such that the at least first nanofiber and
the at least second nanofiber are intertwined. Alternatively, the
first solvent mixture and second solvent mixture may be provided
sequentially one after another such that the at least first
nanofiber and the at least second nanofiber are separate from each
other. In particular, the first solvent mixture and the second
solvent mixture are dispensed to form the at least first nanofiber
and the at least second nanofiber respectively.
[0102] In the preparation of the composite nanofiber construct, in
addition to the solvent mixture comprising a polymer and calcium
salt nanoparticles, higher strength nanofibers with nanofibers
containing less or no calcium salt nanoparticles are electrospun
with the polymer nanofibers containing the desired ratio of polymer
to calcium salt nanoparticles. According to a particular aspect, a
composite nanofiber construct may be formed by depositing at least
a nanofiber layer with the desired mechanical strength to form the
mechanical supporting nanofiber layer, containing less or no
calcium salt nanoparticles (see FIG. 2). Alternatively, the desired
mechanical strength may be achieved by preparing the second
nanofiber having a larger average diameter than the first
nanofiber. A functional nanofiber layer containing the desired
ratio of polymer to calcium salt nanoparticles for enhanced cell
adhesion and attachment is then electrospun onto the mechanical
supporting nanofiber layer as shown in FIG. 2. This bi-layer
composite nanofiber construct consists of a functional composite
nanofiber layer, in which the composition of the nanofiber has been
designed for cell attachment and proliferation, and a mechanical
supporting nanofiber layer, to provide mechanical support for the
construct. In particular, the mechanical supporting nanofiber layer
comprises the at least second nanofiber prepared from the second
solvent mixture and the functional composite nanofiber layer
comprises the at least first nanofiber prepared from the first
solvent mixture. Even more in particular, the second solvent
mixture contains less or no calcium salt nanoparticles.
[0103] The first and second solvent mixtures with calcium salt
nanoparticles and with essentially no calcium salt nanoparticles
respectively, are prepared prior to the electrospinning process.
The calcium salt nanoparticles may be referred to as filler
nanoparticles. These filler nanoparticles are such that they
promote cell attachment, growth and proliferation. The choice of
the filler nanoparticles depends on the cell type desired. For
example, the addition of calcium salt nanoparticles encourages the
attachment and proliferation of osteoblast on the nanofibers.
[0104] The calcium salt nanoparticles are first dispersed in a
particular solvent. This is followed by the addition of the
polymer. The polymer pellet is dissolved in the solvent comprising
the calcium salt nanoparticles. The solvent mixture is mixed using
suitable methods to form the first solvent mixture. For example,
the mixing may be carried out by a magnetic stirrer. Further, a
second solvent mixture may be prepared in a similar manner, except
little or no calcium salt nanoparticles are added to the
solvent.
[0105] According to a particular aspect, the functional composite
nanofibers comprising the at least one first nanofiber are
electrospun simultaneously with the mechanical supporting
nanofibers comprising the at least one second nanofiber, as seen in
FIG. 1B. This results in the formation of a composite nanofiber
construct, in which the functional composite nanofibers are
entwined with the mechanical supporting nanofibers. The
electrospinning apparatus may have two or more dispensers connected
to two or more outlets. By way of example, as shown in FIG. 1B,
dispenser A contains a solvent mixture suitable for electrospinning
mechanical supporting nanofibers comprising the at least second
nanofibers and dispenser B contains a solvent mixture suitable for
electrospinning functional composite nanofibers comprising the at
least first nanofibers. The solvent mixture in dispenser A may
contain little or no calcium salt nanoparticles, while the solvent
mixture in dispenser B may contain a desired ratio of polymer to
calcium salt nanoparticles. The mechanical supporting nanofibers
and the functional composite nanofibers are electrospun
simultaneously to produce entwined nanofibers. The flow rate,
outlet size and polymer concentration can be selected independently
in each of the dispensers to produce nanofibers of the desired
diameter. Further, the composition and diameter of the nanofibers
may be selected to target the growth and proliferation of different
desired cell types. By way of example as shown in FIG. 1B,
dispenser A contains a solvent mixture suitable for electrospinning
functional polymer nanofibers targeted at endothelial cell growth
and dispenser B contains a solvent mixture suitable for
electrospinning functional composite nanofibers targeted at smooth
muscle cells growth. The different functional composite polymer
nanofibers are electrospun simultaneously to produce entwined
nanofibers such that the nanofibers are targeted at growing
multiple cell types on a single construct. The flow rate, outlet
size and polymer concentration may be independently selected in
each of the dispensers to produce nanofibers of the desired
diameter for the growth and proliferation of different cell
types.
[0106] According to another particular aspect, a mechanical
supporting nanofiber layer, comprising the at least second
nanofiber, is first deposited on the collector plate by
electrospinning, followed by the deposition of the functional
composite nanofiber layer, comprising the at least first nanofiber,
by electrospinning as shown in FIG. 2.
[0107] The nanofiber constructs are dried overnight at room
temperature under vacuumed conditions. The morphology of the at
least first and second nanofibers may be influenced by various
processing parameters such as: 1) viscosity of the solvent mixture
determined by the ratio of polymer to calcium salt nanoparticles
and other additives; 2) applied voltage to the electrospinning
apparatus; 3) feed rate of the solvent mixture(s); 4) the distance
between the outlet tip and the collector plate; 5) the inner
diameter of the outlet; and 6) the humidity surrounding the
electrospinning apparatus. Therefore, it is important to optimise
the various parameters to prepare the desired nanofibers.
[0108] According to a further aspect, the method may further
comprise the step of seeding the construct with cells. The cells
may be selected from the group consisting of osteoblasts,
endothelial cells, smooth muscle cells, mesenchymal stem cells,
embryonic stem cells, chrondroblasts, fibrocytes, fibroblasts and
chondrocytes. In particular, the cells are human osteoblasts.
However, any suitable cell type may be used. Further, one type of
cell type or a combination of two or more cell types may be
used.
[0109] According to another further aspect, the method may further
comprise the step of surface functionalizing the construct. The
step of surface functionalising may comprise polymer grafting
and/or plasma treatment. In particular, the step of surface
functionalising comprises plasma treatment of the construct.
Surface functionalization is necessary based on cell type and cell
feature. Surface functionalization of polymer nanofibers includes
polymer grafting, plasma treatment and composite fabrication of
nanofibers. Surface functionalization may also comprise the dipping
and washing of the nanofibers or the construct in sodium hydroxide
solution. Other known methods of surface functionalization suitable
for the purposes of the present invention are also encompassed by
the construct and/or method of the present invention. The advantage
of surface functionalization are as described above.
[0110] As shown in FIG. 3, air-plasma treatment is applied to the
fabricated nanofiber constructs to enhance their hydrophilicity.
Plasma treatment is a useful method to modify the surface-chemical
structure of the constructs to enhance the wettability of the
construct surface. Plasma treatment results in a high-energy
condition producing hydroxyl and carboxyl groups on the surface of
the construct. Plasma-produced polar groups increase the surface
free energy of the construct, resulting in the decrease of the
contact angle. Contact angle is as an estimate of bonding quality.
It is desired to maintain the stability of the plasma treated
construct as the modified surface loses wettability with time. This
is due to the combination of thermodynamical reorientation of polar
groups or the reaction of residual free radicals. In order to avoid
this undesirable loss of surface wettability, the constructs, after
air-plasma treatment, are stored in aqueous solution. According to
a particular aspect, the at least first and/or the at least second
nanofibers are electrospun on 13 mm by 13 mm cover slips. Cover
slips may then be placed on a glass slide, which are placed in a
plasma cleaner chamber. Plasma discharge is applied to the samples
for 10 minutes with the radio frequency power set to 30 W under
vacuum conditions.
[0111] According to another aspect, the present invention provides
a kit comprising the construct of the present invention. The kit
may further comprise a set of instructions on how the construct is
to be used. In particular, the construct in the kit may be an
implant or a scaffold. The kit according to the invention may
further comprise instructions for use(s) and/or application(s) of
the construct, scaffold and/or implant according to the
invention.
[0112] The present invention further provides a method of repairing
fractured bone segments in a subject, comprising the step of
surgically implanting the construct of the present invention. The
subject may be a human or an animal. In particular, the subject is
human.
[0113] The present invention also provides the use of the construct
of the present invention in non-therapeutic and/or cosmetic
applications. It would be known to a skilled person how to use the
construct. For example, the construct may be used in
non-therapeutic and/or cosmetic periodontal purposes. The present
invention therefore provides a method of cosmetic surgery in, but
not limited to, a subject, comprising the step of implanting the
construct according to any aspect of the present invention. In
particular, the method of cosmetic surgery comprises periodontal
surgery.
[0114] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples, which are provided by way of illustration, and are not
intended to be limiting of the present invention.
EXAMPLES
[0115] Standard molecular biology techniques known in the art and
not specifically described were generally followed as described in
Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold
Springs Harbor Laboratory, New York (2001).
Example 1
[0116] In the present invention, the composite nanofiber constructs
were prepared by .epsilon.-polycaprolactone (PCL) nanofibers and a
composite of PCL and calcium carbonate nanoparticles (CaCO.sub.3)
nanofibers with a particular weight ratio, i.e.,
PCL:CaCO.sub.3=25:75 wt %). The materials used were PCL pellet
(Mn=80,000) purchased from Sigma-Aldrich Singapore Pte. Ltd., and
CaCO.sub.3 nanoparticles (average particle size=40 nm: cubic type)
supplied from NanoMaterials Technology Pte. Ltd. Singapore. For PCL
nanofibers, the PCL pellet was first dissolved in a mixture of
solvent comprising 75 wt % chloroform and 25 wt % methanol. The
concentration of PCL solution was 7.5 wt % to ensure fine fiber
morphology in the resulting nanofibers. For PCL/CaCO.sub.3
composite nanofibers, CaCO.sub.3 nanoparticles were first dissolved
in a mixture of solvent and subsequently, the PCL pellet was
dissolved. The concentration of PCL in the resulting mixture was 5
wt %. The outlet was a needle with 0.21 mm inner diameter. The feed
rate of both solvent mixtures were fixed to 1.0 ml/hour by a
dispenser pump. Using a high-voltage power supply (Model: M826,
Gamma High-Voltage Research, USA), 20 kV voltage was applied to the
outlet tip at room temperature and 30%-40% humidity conditions. The
distance between the outlet tip and fiber collector plate was fixed
to 130 mm. The fabricated nonofiber constructs were dried overnight
at room temperature under vacuumed conditionds. FIG. 4 shows a
photograph of PCL nanofibers and PCL/CaCO.sub.3 composite
nanofibers. The average fiber diameter of PCL nanofibers was
600.+-.230 nm while that of PCL/CaCO.sub.3 composite nanofibers was
900.+-.450 nm. FIG. 5 shows the Energy Disperse X-ray (EDX) mapping
of PCL/CaCO.sub.3 composite nanofibers. While the element of
calcium was not recognized on PCL nanofibers (FIG. 5A), the
presence of calcium was detected in composite nanofibers (FIG. 5B).
In present invention, the hydrophilicity of the composite nanofiber
construct was modified by air plasma treatment. Table 1 shows that
10 minutes of air plasma treatment remarkably changed the
wettability of both PCL nanofibers and PCL/CaCO.sub.3 composite
nanofibers. TABLE-US-00001 TABLE 1 Surface contact angle of PCL
nanofibers and PCL/CaCO.sub.3 composite nanofibers before and after
10 minutes of air-plasma treatment. PCL nanofibers PCL/CaCO.sub.3
nanofibers Before 134.degree. 139.degree. After 0.degree.*
0.degree.* *represents that a water drop gradually absorbed into
the mesh.
[0117] After the composite nanofiber constructs were subjected to
air plasma treatment for 10 minutes, osteoblast seeding procedure
was subsequently conducted on the functional composite layer side.
Human osteoblasts (hFOB1.19, catalog no: CRL-11372, ATCC, USA) were
cultured on the composite nanofiber constructs. The cells were
seeded onto 4 samples of scaffolds at a density of 25000
cells/cm.sup.2. The seeding times of the cells were 1, 3 and 5
days. FIG. 6 shows the cell attachment and proliferation manners
observed on the composite nanofiber constructs. The PCL/CaCO.sub.3
composite nanofibers were incorporated with osteoblasts. As seen in
FIG. 6A, granulates, which imply the sign of mineralization
associated with differentiation, were observed on the surface of
the attached cells. It is therefore likely that composite
nanofibers show better bone formation and osteoconductivity under
in vivo conditions.
Example 2
[0118] Please note that with reference to this example, GBR
membrane (A) refers to nanofiber (A) and GBR membrane (B) refers to
nanofiber (B).
2.1 Fabrication of Composite Nanofibrous Construct
2.1.1 Electrospinning
[0119] In this example, composite nanofibrous constructs were
designed by epsilon-polycaprolactone (PCL) nanofibers and
PCL/CaCO.sub.3 composite nanofibers with two different weight
ratios (i.e. PCL:CaCO.sub.3=75:25 wt % and 25:75 wt %). The
materials used were PCL pellet (Mn=80,000) purchased from
Sigma-Aldrich Singapore Pte. Ltd., and CaCO.sub.3 nanoparticles
(average particle size of 40 nm, cubic type) supplied by
NanoMaterials Technology Pte. Ltd. Singapore.
[0120] For PCL nanofibers, the PCL pellet was first dissolved in a
mixture of 75 wt % chloroform and 25 wt % methanol. In order to
obtain fine fiber morphology, the concentration of PCL solution was
varied in the range from 3 wt % to 7.5 wt %.
[0121] For PCL/CaCO.sub.3 composite nanofibers, CaCO.sub.3
nanoparticles were first dissolved in the mixed solvent and
subsequently, the PCL pellet was dissolved. The concentration of
PCL solution was also varied in the range from 3 wt % to 7.5 wt
%.
[0122] As shown in FIG. 1, the prepared PCL solution was placed in
a syringe whose needle inner diameter size was 0.21 mm. The feed
rate of PCL solution was fixed to 1.0 ml/hour by a syringe pump.
Using a high-voltage power supply (Model: M826, Gamma High-Voltage
Research, USA), 20 kV voltage was applied to the needle tip at room
temperature and 30%.about.40% humidity condition. Electrically
charged polymer solution formed a Taylor cone from the tip of the
needle to the ground collector plate with a fixed distance 130 mm.
During this process, the solvent evaporated in the air and randomly
oriented nanofibrous meshes, which were fabricated on the collector
plate. The fabricated samples were dried for one night at room
temperature under vacuumed conditions.
2.1.2 Morphology of Electrospun Nanofibers
[0123] Electrospun nanofibers were coated with gold using sputter
coating and their morphology was observed under scanning electron
microscope (SEM) (Model: JSM-5800LV, JEOL Pte., Ltd.). Energy
Dispersion X-ray (EDX) analysis was also conducted under SEM to
confirm the existence of CaCO.sub.3 nanoparticles on the composite
nanofibers. The average diameter of electrospun nanofibers was
determined by measurement of 30 single nanofibers with the SEM
image using image analysis software (Image J, National Institutes
of Health, USA).
2.1.3 Plasma Treatment
[0124] The nanofibers were air-plasma treated by electrode less
radio frequency glow discharge plasma cleaner (Model: PDC-001,
Harrick Scientific Corporation, USA). The samples were placed on a
glass slide and were stably placed in the chamber of plasma
cleaner. Plasma discharge was applied to the samples for 10 minutes
with the radio frequency power set as 30 W under vacuuming
conditions.
2.1.4 Contact Angle Measurement
[0125] Surface contact angle was measured by contact angle machine
(VCA Optima XE Video Contact Angle System, Crest Technology Pte
Ltd., Singapore). A distilled water drop was put on five different
sites of the nanofibers and the measured angles were averaged.
2.2 Mechanical Characterization of Nanofibers
[0126] The composite nanofibers were carefully cut into the
rectangular dimension of 10 mm width and 60 mm length. Tensile test
of the nanofibers was measured by Instron 5848 Microtester with 10
mm/min cross-head speed with a 40 mm gauge length. Tensile stress
of each membrane was calculated on the nominal cross sectional area
of the tensile specimens (not on total area of nanofibers).
2.3 Osteoblast Proliferation Study
2.3.1 Osteoblast Seeding
[0127] Human osteoblast (hFOB1.19, catalog no: CRL-11372, ATCC,
USA) was cultured on composite nanofibers. Before cell seeding,
sample preparation was conducted as follows. Nanofibers were
fabricated on coverslips whose dimensions were 13 mm by 13 mm and
the edges were stuck together using medical grade silicon adhesive
for one night at room temperature under vacuumed conditions. The
nanofibers on the coverslips were accordingly subjected to plasma
treatment for 10 minutes and transferred to 24-well culture plates.
The nanofibers were then sterilized using 70% ethanol solvent for
60 minutes under UV light and rinsed 3 times by phosphate buffer
saline (PBS). The samples were then incubated in complete medium
which contained Dulbecco's Modified Eagle Medium/Nutrient Mixture
F-12 Ham (DMEM/F12 1:1 mixture: Gibco, USA), 10% fatal calf/bovine
serum (FBS) and 1% penicillin-streptomycin for 12 hours at 34
C..degree. and 5% CO.sub.2. Primary osteoblast culture was
maintained in complete medium until 80% confluency and was passaged
3 times. Osteoblast cells were then detached with 1% trypsin/EDTA
and this was followed by centrifuge and re-suspension processes.
The number of suspended cells were counted using a hemocytometer.
The cells were seeded onto 4 samples of composite nanofibers at a
density of 25000 [cells/cm.sup.2]. The seeding times of cells were
1, 3 and 5 days. At each time, 3 samples were used to measure the
number of attached cells by MTS assay and 1 sample was chemically
treated for SEM observation. For reference purposes, cells were
also seeded to tissue culture polystyrene (TCPS) with the same
seeding conditions.
2.3.2 MTS Assay
[0128] The number of viable cells was measured by MTS assay
(CellTiter 96.RTM. Aqueous Assay). The principle mechanism of this
assay is that metabolically active cells react with a tetrazolium
salt in MTS agent to produce a soluble formazan dye which can be
absorbed at 490 nm wavelength. Each sample was rinsed 3 times with
PBS, followed by incubation with MTS reagent in serum-free culture
medium for 3 hours. Aliquots were then pipetted into 5 wells of a
96-well culture plate and the absorbance at 490 nm of the content
in each well was measured by spectrophotomeric plate reader
(FLUOstar OPTIMA, BMG lab technologies, Germany).
2.3.3 SEM Observation
[0129] In order to observe the cell attachment manner on the
composite nanofibers, chemical fixation of cells was carried out on
each sample. After 1, 3 and 5 days of culture, a sample was rinsed
twice with PBS and subsequently fixed in 2% glutaraldehyde for 1.5
hours. After that, a sample was rinsed with distilled water and
then dehydrated with graded concentration of ethanol, i.e., 50%,
75%, 95% and 100% ethanol for 15 minutes each. Finally, a sample
was treated with hexamethyldisilazane and kept in a fume hood for
air drying. Dried samples were coated with gold using sputter
coating.
2.4 Results
2.4.1 Morphology of Electrospun Nanofibers
[0130] The morphology of polymer nanofibers is influenced by
various processing parameters such as: 1) viscosity of polymer
solution determined by polymer concentration and additives; 2)
applied voltage; 3) feeding rate of polymer solution; 4) distance
between needle tip and collector; 5) needle inner diameter; and 6)
humidity surrounding spinning apparatus. In this study, parameters
except 1) were fixed as follows; 2) voltage=20 kV, 3) feeding
rate=1.0 ml/hour, 4) distance=130 mm, 5) inner diameter=0.21 mm,
and 6) humidity=30.about.40%.
[0131] Viscosity of polymer solution was varied by PCL
concentration and the amount of CaCO.sub.3 nano particles. In terms
of PCL nanofibers, fiber formation was not observed with 3 wt % PCL
concentration. However, increased PCL concentration resulted in
fiber formation with beads, as seen in FIG. 7A. Fine fiber
morphology without beads (see FIG. 7B) was achieved with 7.5 wt %
PCL concentration. The average fiber diameter was 600.+-.230 nm. It
must be noted that beaded nanofibers relatively indicate lower
mechanical strength as compared to fine surface nanofibers. As the
nanofibers require mechanical stability which can sustain surgery
operation, formation of beaded fibers should be avoided. Similarly,
fiber morphology of PCL/CaCO.sub.3 composite nanofibers
(PCL:CaCO.sub.3=25:75 wt %) was also investigated. Although PCL
nanofiber formation was not achieved with 3 wt % concentration,
beaded fibers were fabricated on PCL/CaCO.sub.3 composite
nanofibers with 3 wt % concentration (FIG. 8A). This was due to the
viscosity increase by the addition of 3 times the amount of
nanoparticles against that of PCL. When PCL concentration was
increased to 5 wt %, non-beaded fibers with granulated surface were
formed (FIG. 8B). In order to confirm attached granulation on fiber
surface, Energy Disperse X-ray (EDX) mapping was conducted with
SEM. While the element of calcium was not recognized on PCL
nanofibers (FIG. 5(a)), presence of calcium was detected in
composite nanofibers (FIG. 5(b)). Hence, it was confirmed that
granulation existed on nanofibers when added CaCO.sub.3 nano
particles were added. The average fiber diameter of PCL/CaCO.sub.3
composite nanofibers was 900.+-.450 nm. Based on the
above-mentioned results, PCL/CaCO.sub.3 composite nanofibers with
weight ratio of (PCL:CaCO.sub.3=75:25 wt %) was successfully
fabricated with 7 wt % PCL concentration. The average fiber
diameter was 760.+-.190 nm.
2.4.2 Hydrophilicity of Nanofibers
[0132] The hydrophilicity of the fabricated PCL and PCL/CaCO.sub.3
composite nanofibers were investigated. Table 1 above shows that 10
minutes plasma treatment remarkably changed the wettability of both
PCL and PCL/CaCO.sub.3 composite nanofibers.
2.4.3 Design of Composite Nanofibers
[0133] With this respect, as seen in FIG. 11, the PCL/CaCO.sub.3
composite nanofibers were mechanically supported with PCL
nanofibers which have a higher tensile strength. In this example,
two different types of nanofibers were prepared, i.e., nanofiber
(A): PCL:CaCO.sub.3=75:25 wt %+PCL, nanofiber (B):
PCL:CaCO.sub.3=25:75 wt %+PCL. An equal amount of solution was spun
in each layer of the nanofiber construct. FIG. 12 shows tensile
behavior of nanofiber (B) and the nanofibers could be stretched at
around 200% strain without visible rupture. Nanofibers (A) and (B)
were subjected to plasma treatment for 10 minutes and subsequently
osteoblast seeding procedure was conducted.
2.4.4 Osteoblast Proliferation
[0134] FIG. 9 shows absorbance intensity at 490 nm of TCPS and,
nanofiber (A) (PCL:CaCO.sub.3=75:25 wt %) and nanofiber (B)
(PCL:CaCO.sub.3=25:75 wt %). Absorbance intensity of nanofiber (A)
was similar level to that of TCPS and the values of nanofiber (A)
and TCPS increased during 5 days seeding time. Although absorbance
intensity of nanofiber (B) also increased for 5 days, its value was
lower than the other two samples. As seen in FIG. 10, nanofiber A
had good osteoblast attachment at 1 day (FIG. 10 (a)). The
composite nano fibers incorporated the osteoblast (FIG. 10(b)). For
5 days photo seen in FIG. 10(c), granulates were found on the
surface of a cell. Similar but much more granulations were observed
in nanofiber (B) (FIGS. 10 (d) and (f)) at 1 day and 5 day. It was
also observed that osteoblast incorporated into the composite
nanofibers (FIG. 10 (e)). Although a difference is seen on MTS
assay data between nanofibers (A) and (B), no significant
difference of cell attachment was recognized under SEM observation.
Therefore, when differentiation of osteoblast proceeds,
proliferation rate simultaneously decreases. Hence, there may be a
possibility that because of this osteoblast property, certain
number of cells differentiated on nanofiber (B) which resulted in
lower absorbance than nanofiber (A). Hence, for in vivo condition,
nanofiber (B) may show better bone formation and osteoconductivity
than nanofiber (A).
* * * * *