U.S. patent application number 13/946028 was filed with the patent office on 2013-11-28 for electrospun calcium phosphate nanofibers.
This patent application is currently assigned to Cornell Research Foundation, Inc.. The applicant listed for this patent is Cornell Research Foundation, Inc.. Invention is credited to Yong L. Joo, Jian Tan.
Application Number | 20130313735 13/946028 |
Document ID | / |
Family ID | 39136993 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130313735 |
Kind Code |
A1 |
Tan; Jian ; et al. |
November 28, 2013 |
Electrospun Calcium Phosphate Nanofibers
Abstract
Calcium-phosphate nanofiber matrices comprising randomly
dispersed crystalline calcium-phosphate nanofibers are provided.
The nanofibers are synthesized using sol-gel methods combined with
electrospinning The nanofibers may be hollow, solid or may comprise
a calcium-phosphate shell surrounding a polymer containing inner
core to which biologically functional additives may be added. The
nanofiber matrices may be used to culture bone and dental cells,
and as implants to treat bone, dental or periodontal diseases and
defects.
Inventors: |
Tan; Jian; (Ithaca, NY)
; Joo; Yong L.; (Ithaca, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cornell Research Foundation, Inc. |
Ithaca |
NY |
US |
|
|
Assignee: |
Cornell Research Foundation,
Inc.
Ithaca
NY
|
Family ID: |
39136993 |
Appl. No.: |
13/946028 |
Filed: |
July 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12439398 |
Mar 27, 2009 |
8512741 |
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PCT/US07/77560 |
Sep 4, 2007 |
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13946028 |
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60824377 |
Sep 1, 2006 |
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Current U.S.
Class: |
264/10 |
Current CPC
Class: |
A61L 27/3843 20130101;
A61L 27/50 20130101; C04B 2235/3212 20130101; C04B 30/02 20130101;
C04B 35/63416 20130101; C04B 2235/441 20130101; C12N 2533/18
20130101; C04B 35/62268 20130101; C04B 2111/00836 20130101; A61L
27/12 20130101; A61L 27/3821 20130101; B82Y 5/00 20130101; C12N
2533/30 20130101; B82Y 30/00 20130101; C04B 30/02 20130101; C04B
2235/5284 20130101; C04B 35/447 20130101; C04B 35/62886 20130101;
C04B 2235/447 20130101; C04B 38/0054 20130101; D01D 5/0038
20130101; C04B 35/624 20130101; C04B 30/02 20130101; C04B 35/62881
20130101; A61P 19/00 20180101; C04B 2235/5264 20130101; C12N
2533/40 20130101; C04B 38/0054 20130101; C04B 14/46 20130101; C12N
5/0654 20130101; C04B 20/0056 20130101; C04B 2235/443 20130101;
C04B 35/62849 20130101; D01D 5/003 20130101; Y10T 428/2998
20150115 |
Class at
Publication: |
264/10 |
International
Class: |
D01D 5/00 20060101
D01D005/00; A61L 27/50 20060101 A61L027/50; A61L 27/12 20060101
A61L027/12 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0002] This invention was made with Government support under
contract number NIDCR-DE14097, awarded by the National Institutes
of Health ("NIH"). The Government has certain rights in this
invention.
Claims
1. A method to synthesize a nanofiber, comprising the steps of: (a)
sol-gel synthesizing a calcium phosphate sol-gel precursor in an
aqueous solvent; and (b) electro-spinning the sol-gel precursor to
form a nanofiber.
2. The method of claim 1, further comprising the step of
calcinating the nanofiber produced in step (b).
3. A method to synthesize a nanofiber, comprising the steps of: (a)
sol-gel synthesizing a calcium phosphate sol-gel precursor in an
aqueous solvent; (b) sol-gel synthesizing a polymeric substance
sol-gel precursor in an aqueous solvent; and (c) co-axially
electro-spinning the calcium phosphate sol-gel precursor with the
polymeric substance sol-gel precursor to form a nanofiber, wherein
the nanofiber comprises a calcium phosphate shell around a core
comprising the polymeric substance.
4. The method of claim 3, further comprising the step of
calcinating the nanofiber produced in step (c).
5. The method of claim 1, wherein the calcium phosphate sol-gel
precursor further comprises a polymer binder.
6. The method of claim 1, wherein the sol-gel synthesis of the
calcium phosphate sol-gel precursor in step (a) comprises the steps
of: (i) hydrolyzing a phosphate-based material in an aqueous
solvent; (ii) adding a calcium-based material to the hydrolyzed
phosphate-based material to form a calcium-phosphate sol-gel
precursor solution; (iii) optionally aging the calcium-phosphate
sol-gel precursor solution for at least 16 hours; and (iv)
optionally adding a polymer binder to the calcium-phosphate
solution.
7. The method of claim 6, wherein the calcium phosphate sol-gel
precursor synthesized by said method is a polymer comprising a
plurality of linearly-arranged oligomeric derivatives containing a
Ca--O--P bond.
8. The method of claim 6, wherein the molecular weight of the
calcium phosphate sol-gel precursor is between 10,000 amu and
300,000 amu.
9. The method of claim 8, wherein the molecular weight of the
calcium phosphate sol-gel precursor is between 100,000 amu and
200,000 amu.
10. The method of claim 6, wherein the
Calcium:Phosphate:water:solvent molar ratio is about
1.67:1:3-6.5:7.4-14.8.
11. The method of claim 10, wherein the
Calcium:Phosphate:water:solvent molar ratio is about
1.67:1:6.5:14.8.
12. The method of claim 6, wherein the calcium-based material is
selected from the group consisting of calcium nitrate, calcium
acetate, calcium ethoxide and calcium glycolate.
13. The method of claim 6, wherein the phosphate-based material is
selected from the group consisting of triethyl phosphate and
phosphate ethers.
14. The method of claim 6, wherein the aqueous solvent comprise an
alcohol.
15. The method of claim 14, wherein the alcohol is ethanol.
16. The method of claim 6, wherein the polymer binder is selected
from the group consisting of polyvinyl alcohols, polyglycolic acids
and copolymers thereof
17. The method of claim 16, wherein the polymer binder is added in
the form of a 10% by weight aqueous solution.
18. The method of claim 17, wherein the 10% by weight polymer
binder aqueous solution is mixed with the calcium phosphate sol-gel
precursor at a volumetric ratio between about 1.5 to about 4.5.
19. The method of claim 3, wherein the core-forming polymeric
substance comprises a biodegradable polymer.
20. The method of claim 19, wherein the biodegradable polymer is an
immiscible polymer that does not react or dissolve in ethanol.
21. The method of claim 20, wherein the polymer is selected from
the group consisting of pyridine, poly(glycolic acid), poly(lactic)
acid and poly(lactic acid-co-glycolic acid).
22. The method of claim 20, wherein the polymer is selected from
the group consisting of silica-based compounds.
23. The method of claim 22, wherein the silica-based compound is
tetraethyl orthosilicate (TEOS).
24. The method of claim 3, wherein the sol-gel synthesis of the
polymeric substance precursor in step (b) comprises the steps of:
(ii) catalytically hydrolyzing the polymer in an aqueous solvent;
and (ii) heating the reaction product of step (i), wherein the
sol-gel synthesized polymeric substance precursor comprises at
least one linear chain of oligomer derivatives.
25. The method of claim 2, wherein the calcination step comprises
heating the electrospun fiber to a temperature greater than about
350.degree. C.
26. The method of claim 25, wherein the calcination step comprises
heating the electrospun fibers to a temperature of between about
500.degree. C. and about 1,200.degree. C.
27. The method of claim 26, wherein the calcination step comprises
heating the electrospun fiber to a temperature of between about
600.degree. C. and about 800.degree. C.
28. The method of claim 2, wherein the calcination step comprises
heating the electrospun fiber for between two and twelve hours.
29. The method of claim 28, wherein the calcination step comprises
heating the electrospun fiber for between about five to seven
hours.
30. The method of claim 1, further comprising the step of
evaporating the aqueous solvent.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 12/439,398, filed Mar. 27, 2009, which is a national stage
under 35 U.S.C. .sctn.371 based on and claims benefit of
International Application PCT/US07/77560, filed Sep. 4, 2007,
incorporated by reference, which claims priority to U.S. Prov. App.
Ser. No. 60/824,377 filed on Sep. 1, 2006, the disclosure of which
is hereby incorporated by reference in its entirety.
FIELD OF INVENTION
[0003] The present invention relates to nanofibers and more
particularly nanofiber matrices and nanofiber mats useful in the
regeneration of bone and dental tissue.
BACKGROUND
[0004] Synthetic biomaterials have become increasingly important in
biomedical applications. Calcium-based materials, for example, have
been used for the restoration of bone and dental tissue function.
Various crystalline phases of calcium phosphate ("Ca--P"), such as
hydroxyapatite Ca.sub.5(P0.sub.4).sub.30H, tricalcium phosphate
(Ca.sub.3(P0.sub.4)2, "TCP"), amorphous calcium phosphate
(Ca.sub.3(P0.sub.4).sub.2, "ACP"), octocalcium phosphate
(Ca.sub.8H.sub.2(P0.sub.4).sub.6.5H.sub.20, "OCP"), tetracalcium
phosphate and carbonated or fluoridated apatite have been used in
bone and dental implants in many forms for decades. Dense sintered
hydroxyapatite has been used in middle ear implantation. Calcium
phosphate cement has been used in filling bone defects in dental
and orthopedic surgery. Calcium phosphate coatings on metal
implants have been shown to encourage direct bone deposition on the
implants, thereby forming a strong bond between implants and bone
tissues.
[0005] For repair of bone defects, engineered matrices and
scaffolds should not only provide mechanical support for cell
growth, but should also mimic the extracellular matrix ("ECM") of
the desired tissue. However, current polymeric nanofibers do not
adequately mimic the natural in vivo three-dimensional morphology
and environment experienced by bone and dental cells. Those
polymeric nanofibers that do employ calcium phosphate do so only in
the form of amorphous calcium phosphate mixed into the primarily
polymer based nanofibers. Such polymeric nanofibers do not mimic
the highly crystalline calcium phosphate environment experienced by
bone and dental cells. Moreover, currently existing microfibers are
too large to adequately mimic the ECM of the desired tissue.
However, to date, matrices comprising calcium phosphate nanofibers
in any crystalline phases, particularly those in randomly-dispersed
fibrous networks that mimic the ECM of naturally occurring bone
and/or dental cells, remain elusive. Thus, there is a continuing
need to develop and improve nano-scale fibrous structures
comprising primarily calcium phosphate that can be used for bone
and dental tissue repair and regeneration.
SUMMARY OF INVENTION
[0006] The calcium phosphate ("Ca--P") nanofibers of the present
invention comprise calcium phosphate with one or more Ca--O--P
linkages, such that the phosphate group is selected from the group
consisting of orthophosphate (PO.sup.2-) ions, metaphosphate
(PO.sub.4.sup.3-) ions, and pryophosphate ions
(P.sub.2O.sub.7.sup.4-), and optionally hydrogen or hydroxide ions.
The nanofibers of the invention may take various forms. In one
embodiment, the nanofibers comprise a continuous cross-section of
crystalline calcium phosphate. In another embodiment, the
nanofibers comprise a polymeric core and a crystalline calcium
phosphate shell surrounding at least a portion of the core. In
still another embodiment, the calcium phosphate nanofibers are
hollow (i.e. ,nanotubes). The diameter of the nanofibers may range
from between about 10.0 nm to about 2 microns, preferably between
about 50.0 nm to about 500.0 nm, more preferably between about 75.0
nm to about 300.00 nm, and particularly between about 100.00 and
200.00 nm.
[0007] The nanofibers may exhibit various phases, including
hydroxyapatite (HAP), tricalcium phosphate (TCP, Ca/P=1.5)
tetracalcium phosphate (TTCP, Ca/P=2.0) and octocalcium phosphate
(OCP, Ca/P=1.33). These compounds are typically biocompatible.
[0008] In one embodiment, the present invention is directed to 3-D
nanofiber matrices, mats or scaffolds comprising randomly-dispersed
Ca--P nanofibers with interstices between the nanofibers, such that
the surface area of the nanofiber matrix suitably mimics the
natural environment experienced by bone and dental cells in vivo,
thus providing an adequate environment for bone and dental cells to
grow in both in vivo and in vitro environments.
[0009] The invention is also directed to a method of making 3-D
matrices of calcium-phosphate fibers in which the fibers are
randomly dispersed, as well as to the synthesis of randomly
dispersed calcium-phosphate core-shell nanofibers comprising a
polymeric core and a calcium phosphate shell. The method comprises
the steps of providing (i) a first dispersion comprising at least
one polymeric substance, preferably a linear polymer with an
elongational viscosity between about 1,000 poise and about 3,000
poise; (ii) providing a second dispersion comprising calcium
phosphate; (iii) placing the first dispersion in an inner tube and
the second dispersion in an outer tube of a co-axial syringe; and
(iv) subjecting the dispersion to co-axial electrospinning to
collect a plurality of nanofibers, such that the nanofibers form a
nanofiber matrix of randomly dispersed nanofibers. In some
embodiments, the polymeric core is further seeded with functional
biological additives such as growth factors, cytokines, therapeutic
peptides/proteins, etc., to aid in bone or dental repair or
regeneration.
[0010] The calcium phosphate nanofiber matrices may be used for
various purposes, including use as cell culture systems, cell
carriers, and the repair of various body parts and organs, such as
bone and dental tissue repair/regeneration and skin grafts.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Certain embodiments of the present invention are illustrated
by the accompanying figures. It should be understood that the
figures are not necessarily to scale and that details not necessary
for an understanding of the invention or that render other details
difficult to perceive may be omitted. It should be understood, of
course, that the invention is not necessarily limited to the
particular embodiments illustrated herein.
[0012] FIG. 1 is a scanning electron microscope ("SEM") image of a
nanofiber mat made in accordance with one embodiment of the present
invention;
[0013] FIG. 2 is a perspective view of an electrospinning apparatus
used to prepare the nanofibers of the present invention;
[0014] FIG. 3 is a perspective view of an alternate setup of the
electrospinning apparatus of FIG. 2, adapted for co-axial
electrospinning;
[0015] FIG. 4 illustrates a flow-chart of the general parameters
for the sol- gel process of the present invention with an
accompanying viscosity curve;
[0016] FIG. 5 illustrates the growth of osteoblast-like Saos-2
(Saos-2) cells on a nanofiber mat comprising co-axial fibers made
in accordance with one embodiment of the present invention. The
fibers comprised a silica core and a calcium phosphate shell;
and
[0017] FIG. 6 illustrates live DPM cells stained with fluorescence
to show that about 90% of the cells were alive after a week-long
culture on the nanofiber mat of the present invention.
DETAILED DESCRIPTION
[0018] Non-woven nanofibers of the present invention comprise a
plurality of repeating calcium phosphate units. In certain
embodiments, the nanofibers comprise substantially non-derivatized
calcium phosphate (i.e., the chemical structure of the calcium
phosphate is void of other elements, compounds and substances
except for impurities). In other embodiments of the invention, the
calcium phosphate is either substantially crystalline, or amorphous
with some crystalline structure. The term calcium phosphate, as
used herein, means the family of compounds containing calcium ions
(Ca.sup.2+) together with at least one of orthophosphate ions
(PO.sup.2-), metaphosphate ions (PO.sub.4.sup.3), or pryophosphate
ions (P.sub.2O.sub.7.sup.4-), and optionally hydrogen or hydroxide
ions, having one or more Ca--O--P linkages. Examples include
tricalcium phosphate Ca.sub.3(P0.sub.4).sub.2 (also called tribasic
calcium phosphate, occurring in alpha and beta phases), dicalcium
phosphate CaHP0.sub.4 (also called calcium monohydrogen phosphate),
calcium dihydrogen phosphate Ca(H.sub.2P0.sub.4).sub.2 (also called
monocalcium phosphate) and hydroxyapatite
Ca.sub.10(P0.sub.4).sub.6(OH).sub.2. A scanning electron microscope
image of a plurality of nanofibers according to one embodiment of
the present invention is shown in FIG. 1. When the plurality of
nanofibers is randomly dispersed, with at least some of the
nanofibers in physical contact with one another, a nanofiber
matrix, or mat, is formed. The nanofiber mat may contain
interstices, gaps or pores between the nanofibers.
[0019] The nanofibers may also comprise a polymer binder. The
polymer binder may include linear aliphatic polymers often used in
tissue engineering including poly(glycolic acid) ("PGA"),
poly(lactic) acid ("PLA," which by virtue of the extra methyl group
in its repeating unit is more hydrophobic than PGA), and their
copolymers such as poly(lactic acid-co-glycolic acid) ("PLGA"). The
properties of the PLGAs may be varied by altering the ratio of
lactic acids to glycolic acids. These polymers (PLA, PGA, and
PLGAs) are also preferred as they have been approved by the U.S.
Food and Drug Administration ("FDA") for human clinical
applications.
[0020] Other biocompatible linear aliphatic polymers include
polyethers such as polyethylene glycol ("PEG"), polyethylene oxide
("PEO"), polypropylene oxide ("PEP") and their co-polymers, for
instance, poly(ethylene glycol-co-propylene oxide) ("PEG-PPO").
Suitable polymer binders also include poly(-caprolactone) ("PCL"),
poly(hydroxy butyrate) ("PHB") and their respective copolymers. In
another embodiment, biodegradable polymers including poly(propylene
fumarate) ("PPF"), tyrosine-derived polymers, segmented
polyurethane-based polymers, polyphosphoesters, polyphosphazenes,
polyanhydrides and poly(ortho esters) may be used as polymer
binders. In another embodiment, the polymer binder may be a
water-soluble vinyl polymer. Water-soluble vinyl polymers include
polyvinyl alcohol ("PVA"), polyvinyl ether and polyvinyl
pyrrolidone. In yet another embodiment, block co-polymers
comprising combinations of two or more of any of the aforementioned
polymers may be used.
[0021] In one embodiment, the nanofiber may be solid when seen in
any cross-section to the longitudinal axis of the nanofiber, such
that the nanofiber comprises a continuous cross-section of calcium
phosphate. The cross-section may be taken along a perpendicular
axis. In another embodiment, the nanofiber may comprise a calcium
phosphate shell surrounding a hollow core. In yet another
embodiment, the calcium phosphate shell surrounds a solid core
having at least one polymeric substance. The polymeric substance
present in the core may comprise one or more biodegradable
polymers, alone or in combination with other functional
additives.
[0022] The calcium phosphate nanofibers exhibit various
advantageous properties, cross-sectional distances (or diameters
when the cross-section of the nanofibers closely approximates a
circle) in the nanometer range, a three-dimensional network
structure, and interconnected porosity. The nanofibers are
relatively thin, comprising cross-sectional dimensions ranging in
size from about 10.0 nm to about 2.0 microns when cut or viewed in
cross section which may be taken perpendicular to the longitudinal
axis of the fiber. In another embodiment, the cross-sectional
dimensions may range from about 50.0 nm to about 500.0 nm, while in
a third embodiment, the cross sectional dimensions range from about
75.0 nm to about 300.0 nm. In a fourth embodiment, the cross
sectional dimensions of the nanofiber are from about 100.0 nm to
about 200.0 nm. In those instances, where the cross-section of the
fiber approximates a circle, the cross-sectional dimension is the
diameter of the circle. In other instances where the cross-section
is irregularly-shaped, the cross-sectional dimension is the maximum
cross-sectional distance between any two points on the surface of
the nanofiber taken on a cross-section perpendicular to the
longitudinal axis of the nanofiber.
[0023] Fiber cross-sectional distances, such as fiber diameters,
are measured using scanning electron microscopy (e.g., Zeiss Supra
or Ultra SEM with a resolution of 1 nm). In order to directly probe
the Ca--P crystals with 1.0 nm resolution in nanofibers, elemental
mapping on an Energy Filtering Transmission Electron Microscope
("EFTEM") using electron energy loss spectroscopy ("EELS") is
utilized. The surface charge of the nanofibers is typically neutral
to net positive, as determined by zeta potential testing
[0024] The nanofibers may possess a plurality of internal pores.
The dimensions of the pore range from about 0.1 nm to about 10.0
nm. In another embodiment, the size of internal pore ranges from
about 2.0 nm to about 5.0 nm. In one embodiment of the invention, a
plurality of internal pores may interconnected.
[0025] As previously stated, the nanofibers may exhibit various
phases, including hydroxyapatite ("HAP"), tricalcium phosphate
("TCP," Ca/P=1.5 (molar ratio "m/m")), tetracalcium phosphate
("TTCP," Ca/P=2.0 (m/m)) and octocalcium phosphate ("OCP,"
Ca/P=1.33 (m/m)). The crystal habit of these compounds ranges from
monoclinic to triclinic to hexagonal. X-ray diffraction ("XRD") is
used to probe the presence of calcium phosphate crystals and
determine structural characterization to compare with standard
calcium phosphate phases such as HAP, OCP, TCP, etc. The degree of
crystallinity may be at least about 40% and preferably between
about 70% and 99%, the non-crystalline calcium phosphate being
amorphous. The degree of crystallinity can be determined by XRD,
calorimetry, density measurements or infrared spectroscopy.
[0026] The mechanical strength of nanofibers may also be
characterized. Tensile strength and compressive strength may be
between about 25.0 and 1,000.0 MPa (millipascal). Tensile
experiments of bulk nanofiber mats are carried out on a
conventional Instron 1125 test system (www.instron.com), while the
modulus and yield strength of a single Ca--P nanofiber may be
measured by depressing the suspended nanofiber with a tip of an
Atomic Force Microscope.
[0027] A plurality of nanofibers, randomly dispersed, may take the
form of a three-dimensional nanofiber matrix. Such a matrix may
alternatively be termed a scaffold or a mat. When the nanofibers
take the form of a nanofiber mat, the mat may exhibit a relatively
high specific surface area, owing to relatively small nanofiber
diameters and the presence of porosity as a result of pore-like
interstices within the nanofiber mat. The specific surface area of
the nanofiber mat, measured with a Micrometrics Phys/Chemi Sorption
Analyzer (http://www.micromeritics.com/), is typically greater than
about 10 m.sup.2/g and more particularly between about 100
m.sup.2/g and 1,200 m.sup.2/g or 600 m.sup.2/g and 1,100 m.sup.2/g,
and still more particularly between about 800 m.sup.2/g and about
1,000 m.sup.2/g. The nanofiber mat may include inter-fiber pores.
Such pores within the mat are defined by the tiny interstices or
gaps between the randomly dispersed nanofibers of the mat. The size
of the pores between the randomly-dispersed nanofibers range from
about 0.5 microns to about 50.0 microns, more particularly between
about 1.0 micron to about 10.0 microns and may be measured using
scanning electron microscopy.
[0028] To make the nanofibers of the present invention, various
processes may be employed. In one embodiment, calcium phosphate
nanofibers may be made by electrospinning a sol-gel precursor
comprising calcium phosphate and a polymer binder, followed by
optional calcination, a thermal treatment process by which a
volatile fraction, such as a polymer binder, water or solvent may
be removed. In another embodiment, nanofibers may be made by the
same process without the use of a polymer binder. Additionally,
nanofibers comprising a calcium phosphate skin layer (or shell) and
a biodegradable polymeric core may be made by employing co-axial
electrospinning.
[0029] The goal of the sol-gel synthesis is to preferably yield a
reaction product comprising a relatively high elongational
viscosity, for example between about 10.0 poise to about 1,000.0
poise. If the solution viscosity is too low, either beads of
solution will form on the fibers or only discontinuous jets via the
electrospinning mechanism will be formed. If the viscosity of the
solution is too high, fiber yield may be compromised. Preferably,
the solution viscosity is between about 10.0 poise and 1000.0
poise, more particularly between about 200.0 poise to about 600.0
poise.
[0030] A schematic of the sol-gel synthesis is shown in FIG. 4. To
carry out the sol-gel synthesis, calcium and phosphorous based
starting materials are combined. Suitable calcium-based starting
materials include, but are not limited to, materials comprising
calcium nitrate, calcium acetate, calcium ethoxide and calcium
glycolate; suitable phosphate-based starting materials include, but
are not limited to, materials comprising triethyl phosphate, and
various phosphate.
[0031] An illustrative embodiment for synthesis of Ca--P sol-gel
precursor as shown in FIG. 4 involves triethyl phosphate 402 and
calcium nitrate tetrahydrate 400. This synthesis begins with the
addition of triethyl phosphate 402 to anhydrous ethanol 404 in a
vial. Distilled water 406 is then added and the solution is allowed
to hydrolyze for at least about 1.0 hour with vigorous stirring.
Calcium nitrate tetrahydrate 400 dissolved in anhydrous ethanol 404
is added dropwise to the triethyl phosphate solution while stirring
to form the sol-gel precursor 408. To achieve the appropriate
elongational viscosity, the molar ratio of starting material to
solvent to water to catalyst may be adjusted. When the solvent used
is ethanol, the molar ratios of Ca:P:water:ethanol may be about
1.67:1:3-6.5:7.4-14.8, with a preferred ratio of 1.67:1:6.5: 14.8.
An aging process 420 follows in which the solution is aged for at
least about 16.0 hours and more particularly between 16.0 hours and
72.0 hours. After aging, an evaporation process 430 follows, in
which the solvents are evaporated at temperatures ranging from
25.degree. C. to about 80.degree. C. for about 6.0 hours to obtain
a clear viscous spinnable liquid 412. A solution of 10% by weight
of a polymer binder and water 414 is optionally mixed with the
calcium phosphate solution in a volumetric ratio of about 9:1 or
8:2, 7:3 and 6:4. The polymer binder may be polyvinyl alcohol
("PVA") or polyglycolic acid. After evaporation of solvents at
80.degree. C., the calcium phosphate sol is stirred for about an
hour to obtain a relatively homogeneous mixture.
[0032] The sol-gel synthesis of calcium phosphate proceeds as
follows:
P(OEt).sub.3-x(OH).sub.x+Ca(N0.sub.3).sub.2-y(OEt).sub.y.fwdarw.
(OET).sub.y'(N0.sub.3).sub.2-y'Ca--O--HPO(OEt).sub.3-x'+H.sub.20+Ca.sub.-
2H.sub.20H
Hydrolyzed phosphorus sol in the form of phosphoric ester
P(OEt).sub.3-x(OH).sub.x with Ca in the form of
Ca(N03).sub.2-y(OEt).sub.y in anhydrous ethanol may be used to form
Ca--P sol comprising oligomeric derivatives linked by Ca-0-P bonds.
Ca-0-P bonds provide a linear structure suitable for obtaining
fibers via electrospinning. The schematic of the calcium phosphate
nanofiber scaffold fabrication via electrospinning is shown in FIG.
2. The ratio of water, ethanol, calcium, and phosphorous can be
varied to produce fibers with desirable features. It is understood
that determination of the ratios may be made by one of ordinary
skill in the art without undue experimentation. PVA may be added to
the Ca--P sol-gel precursor to enhance processability and to aid in
the production of substantially continuous fibers.
[0033] The ultimate reaction product of the sol-gel synthesis may,
thus, comprise a linear polymer with the general formula F:
##STR00001##
Wherein X=calcium Ca, Y=phosphorus, P and R.sub.1-R.sub.8.noteq.
oxygen bonded to another one of X or Y. Typically,
R.sub.1-R.sub.8.dbd.OCH.sub.2CH.sub.3. Such a configuration helps
optimize nanofiber formation during the electrospinning step,
avoiding unwanted break-down or disintegration of material. The
molecular weight of the linear polymer is typically between about
10,000 amu and 300,000 amu and more particularly between about
100,000 amu to 200,000 amu. The control of elongational viscosity
in electrospinning is more important than in conventional spinning
because of higher elongational deformation during
electrospinning.
[0034] In the event co-axial electrospinning of a calcium phosphate
shell on a chemically distinct polymeric core is desired, other
types of sol-gel synthesis may be employed to produce a sol-gel
precursor to the polymeric core. Virtually any immiscible polymer
that does not react or dissolve in ethanol may be employed in the
polymeric core. Pyridine, polylactic co-glycolic acid and
tetraethyl orthosilicate ("TEOS") are just some suitable
examples.
[0035] A sol-gel synthesis reaction using TEOS as a precursor is
illustrative. TEOS is added to a solvent of ethanol and water,
followed by the dropwise addition of a catalyst (for example, a
solution of hydrochloric acid in water), with vigorous stirring. In
one embodiment, the molar ratios of TEOS: EtOH: H20: HCL are about
1:2:2:0.01. Although organic polymeric binders, such as PVA may be
employed, the reaction may proceed without the use of such binders.
The solution is heated for one to three hours at a temperature of
about 50.degree. C. The aforementioned steps yields a dispersion
comprising at least one linear chain with the general formula:
##STR00002##
[0036] Various additives may also be incorporated into the
above-described sol-gel precursor. In one embodiment, such
additives include compounds that improve cell function and
mechanical strength of the electrospun fibers, for instance,
compounds containing carbonate and fluoride ions to make carbonated
or fluorinated apitite. Compounds containing these ions are
typically added to the sol, with fluoride added in an amount less
than about 5.0 wt. %, whereas carbonate may be added in an amount
less than about 20.0 wt. %. Other additives, such as compounds
comprising Sr, Mg, Mn, Zn, Na and K, may also be employed.
Additionally, biologically active molecules, such as proteins,
peptides, DNA, RNA, antibiotics, antimicrobials,
anti-inflammatories, steroids and chemotherapeutical agents.
Examples include osteogenic factors, nuerotrophic factors,
angiogenic factors, bone morphogenic protein ("BMP"), transforming
growth factors ("TGF"), vascular endothelial growth factor
("VEGF"), platelet-derived growth factor ("PDGF"), neurotrophins,
cytokines, etc. The presence of these materials in the sol yields a
co-axially electrospun core comprising these same advantageous
materials. Biphasic calcium phosphate ceramics are also possible
through the methods of the present invention.
[0037] After the sol-gel step and recovery of the dispersion
comprising linear chains, the dispersion is subjected to
electrospinning. Electrospinning is a fiber formation process that
relies on electrical, rather than mechanical forces to form thin
fibers with diameters ranging between about 50.0 nm and about 10.0
microns. A strong electric field is used to draw a solution from
the tip of a capillary to a grounded collector. The electric field
causes a pendant droplet of the solution at the capillary tip to
deform into a conical shape. When the electrical force at the
surface of the tip overcomes the surface tension of the solution, a
charged jet is ejected. The jet moves toward the collector plate,
which acts as a counter electrode. The solvent begins to evaporate
after jet formation, causing the deposit of a thin fiber on the
collector. To the extent any solvent remains, the fibers may be
heated at temperatures of about 150.degree. C. to remove residual
solvent.
[0038] Referring now to FIG. 2, one embodiment of an
electrospinning apparatus 100 for use with the present invention is
illustrated. Apparatus 100 comprises syringe 102, comprising an
inner diameter of between about 0.20 millimeters to about 0.60
millimeters, tip 104, high voltage supplier 106 positioned at or
near tip 104, and collection plate 108, constructed of a conductive
material, such as aluminum, stainless steel, or a surface oxidized
silicon. The diameter of nanofibers may be decreased by decreasing
the inner diameter of syringe 102. The distance between tip 104 and
collector 108 is about 10.5 centimeters. High voltage supplier 106
includes a voltage of about 20 kV. Collector 108 is grounded to
create an electric field difference between tip 104 and collector
108, causing jet 110 to move from the high electric field at tip
104, to grounded collector 108. Collector 108 may also be a
rotating collector.
[0039] Once apparatus 100 is assembled, dispersion 109 created
during the sol-gel synthesis step is placed into syringe 102, and
pumped through at a relatively constant flow rate of about 0.03 mL
per minute. As pumping continues, charged jet 110 is ejected and
elongates as it moves towards collector 108. Thus, a plurality of
randomly oriented non-woven ultra-thin fibers or nanofibers 112 are
collected on collector 108. At this point, the nanofibers 112
comprise a three-dimensional network of calcium phosphate.
[0040] In an another embodiment, shown in FIG. 3, co-axial
electrospinning may be employed. In co-axial electrospinning, a
dual syringe pump with controlled flow rates of both inner and
outer solutions is used.
[0041] Co-axial electrospinning apparatus 200 comprises dual
syringe 202 which comprises an internal tube positioned within an
external tube, tip 204, high voltage supplier 206 and collector 208
for receiving nanofibers 212. Under this construction an internal
jet with an external jet is ejected from the syringe; the internal
jet may comprise polymeric substances such as those prepared from
TEOS, while the external jet comprises the calcium phosphate
dispersion 209, prepared during sol-gel synthesis.
[0042] Alternatively, the sol-gel dispersion may be subjected to
conventional mechanical spinning using an extruder, die and winder.
Conventional spinning employs a mechanical force to draw fibers out
of solution and yields fiber diameters between about 3.0 microns
and about 5.0 microns.
[0043] After electrospinning, the polymeric binder may be removed
from the fibers through calcination. Calcination involves heating
to relatively high temperatures. The calcination temperature and
time depend upon the materials being used as well as the needs of
the user. The typical temperature ranges from 500.degree. C. to
about 1,200.degree. C. and more particularly from about 600.degree.
C. to about 800.degree. C. Calcination is typically carried out
anywhere between two and twelve hours and more particularly between
about five to seven hours. Through the calcination process, the
polymer binder is effectively removed and the resulting fibers
comprise essentially calcium phosphate.
[0044] Calcination also impacts several properties of the
nanofibers. Calcination can decrease nanofiber diameters and
internal porosity and transform amorphous materials into crystals.
Higher temperatures produce nanofibers with smaller diameters. In
addition, extended calcination typically decreases and ultimately
removes the internal porosity of the fibers. Provided calcination
does not proceed too far, the collective nanofibers themselves (as
opposed to the mat) may comprise a plurality of internal pores, at
least one of which exhibits a diameter between about 0.1 nm and
about 10.0 nm and more particularly between about 2.0 nm and about
5.0 nm.
[0045] If co-axial electrospinning is employed, the calcining step
may be eliminated, or its duration and/or temperature may be
reduced, as calcination may lead to removal of the polymeric core.
In order to maintain the presence of the core, therefore, the
collected nanofibers are preferably not exposed to calcination
temperatures for extended periods. Such co-axial electrospinning,
however, may rely on the presence of various polymer binders added
to the Ca--P sol, such as polylactide, poly
(D,L-lactide-co-glycolidepolylactide) and/or polycaprolactone in
methylene chloride. In order to remove the polymer binders while
leaving the polymeric cores intact, calcination may be controlled
by changing the temperatures and time of calcination.
[0046] The final product generated by the above-described three
step process is a nanofiber mat comprising a plurality of
nanofibers comprising non-derivatized crystalline calcium
phosphate. Some nanofibers within the mat may be adhered to one
another, based on the presence of residual solvent. The nanofibers
may comprise a three-dimensional network. To increase crystal
content on external surfaces, the fibers may be placed in water for
0.5 hours to about 1 hour prior to calcination. This step drives a
hydrolysis reaction, which ultimately increases crystal growth by
between about 15% to about 50% of the original coverage.
[0047] The calcium phosphate nanofibers may be used for various
purposes, including repair of various body parts and organs, such
as bone and dental tissue repair/regeneration and skin grafts. For
example, calcium phosphate nanofiber mats be implanted during
surgery or electrospun onto the surface of a soft tendon tissue so
that the tendon can directly connect with hard bone to reconstruct
an injured ligament implanted during surgery. The calcium phosphate
fibers also have potential applications in dental tissue repair and
regeneration (in particular guide tissue regeneration ("GTR")) of
dental structures, such as dental tissues, damaged by dental or
periodontal diseases. The calcium phosphate fibers also have
potential applications in repair and regeneration (in particular
guide tissue regeneration ("GTR")) of bone tissues, damaged by
injury, disease or as a result of genetically-based bone defects.
To this effect, the calcium-phosphate nanofiber mats assist in
bone/dental tissue engineering by serving as cell carriers. In such
instances, the calcium-phosphate fiber mats are used as bone or
dental implants in which the mats are cultured with the necessary
bone or dental cells, respectively, in vitro such that the cells
adhere to the mat and reach tissue-like densities, and then
implanted adjacent to the damaged or diseased bone or dental
tissue, or to fill bone and dental tissue defects. Such bone and
dental cells include osteoblast and odontoblast cells respectively.
The fibers are also useful in reconstructing an injured ligament in
between hard bone and soft tendon tissue, by implanting the
nanofiber matrix onto the surface of the soft tendon tissue and
adjacent to the injured ligament such that adjacent bone and tissue
can proliferate on the matrix, allowing the tendon to connect to
the hard bone.
[0048] As the cells show microstructure dependent behavior, it is
believed that adhesion, proliferation and differentiation of the
cells is significantly improved through use of the present
invention. The fiber network call be seeded or cultured with such
cells before implantation. SEM images of co-axially electrospun
fibers, comprising a silica core and a calcium phosphate shell, are
shown in FIG. 5. These fibers were seeded with Saos-2 cells, which
penetrated the fiber network and exhibited a substantially round
morphology similar to the these types of cells in vivo. As shown in
FIGS. 5 and 6, these cells grow well in the presence of the
nanofibers of the present invention.
[0049] Additionally, core-shell nanofibers are useful for the
delivery of biofunctional molecules such as growth factors to
enhance guide tissue regeneration. Core-shell fibers therefore
enable a novel method of delivery of biofunctional molecules.
[0050] The present invention is illustrated, but in no way limited
by the following examples.
EXAMPLES
[0051] In Examples 1 (a) and 1 (b), calcium phosphate nanofiber
mats were generated using sol-gel synthesis with a polymer binder,
followed by electrospinning and calcination.
Example 1 (a)
[0052] Triethyl phosphite and calcium nitrate were selected as
phosphate and calcium precursors for nanofiber synthesis of
hydroxyapatite nanofibers. To produce such fibers, a molar ratio of
calcium to phosphate of 1.67: 1 was desirable. The resulting Ca: P:
water: ethanol mole ratio for this study was 1.67: 1: 6.5: 14.8.
Triethyl phosphite (ETO).sub.3P (MP Biomedicals) was added to 2.0
mL of 200-proof ethanol in a small vial. Distilled water in an
amount of 1.35 mL was subsequently added to maintain a 3.0 M
phosphorus precursor solution. The phosphite solution was then
allowed to hydrolyze for 1 hour at room temperature with vigorous
stirring. In a separate small vial, calcium solution was prepared
by dissolving 4.935 g of calcium nitrate tetrahydrate
Ca(N0.sub.3).sub.2.4H.sub.20 (J. T. Baker) in 7 mL of 200-proof
Dissolution of the calcium compound was facilitated by vigorous
stirring at room temperature for 30 minutes. The final volume was
approximately 7.5 mL. Upon completion of the phosphite hydrolysis,
a stoichiometric amount of the calcium nitrate solution was added
dropwise into the hydrolyzed phosphite sol. Vigorous stirring was
continued for 15 minutes to obtain a clear solution that was left
to age at room temperature for at least 16 hours prior to use.
[0053] A polyvinyl alcohol (PVA, Polyscience, Inc., MW 78,000, 88
mole % hydrolyzed) solution was also prepared for incorporation
into the calcium phosphate solution as a polymer binder. Initially,
a 10% w/v PVA/water solution was prepared by addition of polyvinyl
alcohol to water. This solution was stirred, covered, at room
temperature (about 25.degree. C.) until the majority of the PVA
dissolved. The solution was then heated to 80.degree. C. and
stirred for an additional 30-60 minutes until all remaining PVA
particles dissolved. The final 1.425*10.sup.-3 M PVA solution was
clear, viscous, and easily spinnable.
[0054] A 4:6 volumetric ratio Ca--P sol-gel precursor/PVA solution
was prepared prior to electrospinning by adding 10% w/v PVA to the
Ca--P sol dropwise under vigorous stirring. In order to achieve a
viscosity suitable for electrospinning, this solution was then
stirred open to ambient air for at least 24 hours to evaporate
volatile solvents. The sol-gel precursor was allowed to settle for
at least 24 hours to achieve the best spinning results.
[0055] The electrospinning conditions employed were as follows:
distance from tip to collector=0.127 meters, Q=0.001 mL/min,
Voltage=19-20 kV, time=2 hours. The as-spun fibers were transferred
to a 50.degree. C. thermal convection oven and stored for at least
48 hours to facilitate solvent evaporation prior to calcination.
Solvent evaporation during this step allowed formation of intact
fiber mats; as-spun fibers that were calcined without adequate
solvent removal tended to merge together into films upon exposure
to ambient air.
[0056] The as-spun fiber samples were placed in a ceramic boat and
calcined in a box furnace in air. The calcination temperature was
chose to: 1) promote hydroxyapatite formation from the calcium
phosphate precursors (hydroxyapatite was shown to form from this
Ca--P sol-gel precursor at temperatures at and above 350.degree.
C.) and 2) completely remove the polymer binder, PVA, from the
fiber sample. The calcination conditions employed were: ramp
rate=800.degree. C./hour; calcination temperature=400.degree. C.;
duration=3 hours; rate of decrease in temperature=2000.degree.
C./hour; and final temperature=room temperature.
Example 1(b)
[0057] In Example 1 (b), various polymeric core/Ca--P shell
nanofibers were produced by electrospinning different sol-gel
precursor solutions through a spinneret comprised of two coaxial
capillaries. These nanofibers included:
[0058] Type 1. Shell: non-derivatized calcium phosphate [0059]
Core: silica-containing polymeric substance
[0060] Type 2. Shell: calcium phosphate +PVA [0061] Core:
silica-containing polymeric substance
[0062] Type 3. Shell: silica-containing polymeric substance [0063]
Core: non-derivatized calcium phosphate
[0064] In all cases, the core (inner solution) and shell (outer
solution) solutions for electrospinning were sol-gel precursors of
the respective materials. During electrospinning, the solvent began
to evaporate from both solutions, thus promoting the transition
from the sol to gel state. Calcination of as-spun fibers was
performed in order to produce fibers as well as to obtain the
desired hydroxyapatitic crystalline structure.
[0065] Silica was chosen as a material for both core and shell
because of the ease of its preparation via the sol-gel method and
also because of the ease of its electrospinning. It was thought
that the sol-gel transition of silica sol-gel precursor during
electrospinning could be harnessed to drive the formation of the
calcium component of the coaxial nanofibers.
[0066] The silica sol was prepared from tetraethyl orthosilicate
("TEOS"), distilled water, ethanol, and hydrochloric acid in a
molar ratio of about 1:2:2:0.01. First, TEOS was combined with
ethanol in a beaker. The HCl water solution was then added dropwise
to the TEOS/ethanol mixture under vigorous stirring to obtain a
clear, immiscible sol. This solution was placed in a 50.degree. C.
thermal convection oven for 4-5 hours; solvent evaporation during
this time period produced a clear sol with a viscosity suitable for
electrospinning.
[0067] The calcium phosphate sol was prepared as outlined in
Example 1 (a). In Type 1 and 3 fibers, this sol was evaporated to a
viscosity suitable for electrospinning under vigorous stirring at
80.degree. C.; evaporation times varied depending on the surface
area of sol exposed to ambient conditions. In Type 2 fibers, a 4:6
volumetric ratio calcium phosphate sol-gel precursor/PVA solution
was prepared prior to electrospinning by adding 10 wt. % PVA to the
calcium phosphate sol dropwise under vigorous stirring. In order to
achieve a viscosity suitable for electrospinning, this solution was
then stirred open to ambient air for at least 24 hours to evaporate
volatile solvents. The Ca--P/PVA sol-gel precursor was allowed to
settle for at least 24 hours to achieve the best electrospinning
results.
[0068] Co-axial electrospinning was carried out under the following
conditions
TABLE-US-00001 Type 1 Type 2 Type 3 Shell: pure Shell: Ca--P/PVA
Shell: silica Ca--P Core: silica Core: silica Core: Ca--P TCD*
(in.) 4-6 5 5 Q.sub.in (mL/min) 0.02-0.03 0.02 0.02 Q.sub.out
(mL/min) 0.002-0.005 0.005 0.005 v (kV) 20 23 23 t (hr) 2 2 2 TCD =
tip-to-collector distance Q.sub.in = volumetric flowrate of core
solution Q.sub.out = volumetric flowrate of shell solution V =
voltage t = duration of electrospinning
[0069] As-spun fibers were stored in a 50.degree. C. thermal
convection oven to promote solvent evaporation prior to
calcination.
[0070] The as-spun fibers were placed in a ceramic boat and
calcined in a box furnace in air. The calcination temperature was
chosen primarily to promote hydroxyapatite formation from the
calcium-phosphate precursors (hydroxyapatite was shown to form from
this sol-gel precursor at temperatures at and above 350.degree. C.
In Type 2 fibers, calcination also allowed removal of the polymer
binder, PVA, from the fiber sample. The calcination conditions were
the sane as those described above in connection with Example
1a).
Example 2
[0071] The stability and resorbability of the nanofibers of the
invention in cell culture medium was studied to determine if the
nano fibers undergo physical and chemical modification in a cell
culture medium. The stability (or resorbability) of the nanofibers
in the medium is studied first without the cells. Nanofibers were
removed from the medium at various time points and observed using
XRD and SEM for structural changes. The information is helpful to
understand cell behavior; to determine the ratio of the rate of
cell differentiation to cell proliferation; to evaluate protein
adsorption, membrane receptor/ligand interactions, cytoskeleton
organization and subsequent signal transduction; and to correlate
in vitro studies to animal models.
[0072] To test biocompatibility and obtain optimal structures for
in vivo evaluation of the fibrous structures in bone and dental
tissue regeneration, the influence of the following parameters on
bone and dental cell behavior were also investigated: chemical
composition (Ca/P ratio), crystalline structure, crystallinity,
fiber diameter, pore size, stability and mechanical strength.
[0073] Two model cell lines were used for an in vitro test of
biocompatibility: osteoblast-like Saos-2 and dental papillae
mesenchyme ("DPM") cells (www.atcc.org), an immortal odontogenic
cell line. Saos-2 cells possess osteoblastic characteristics and
were used as model cells to test compatibility with the nanofibers
for bone regeneration and dental repair, especially when used as a
coating of dental metal implants or as a membrane for guide tissue
regeneration of periodontal and dental tissues (periodontal
membrane, alveolar--bone, cementium and dentin) weakened or damaged
by related diseases. DPM cells derived from Immortomouse, a
transgenic mouse harboring the SV40 strain A58 early region coding
sequences under the control of the mouse MHC H-2Kb class I promoter
(http://www.criver.com/research_models_and_services/transgenic_s-
ervices/tgresearchmo dels.html), are used. These transgenic mice
(H-2Kb-tsA58) have the potential of producing cell lines derived
from many different tissues. For example, periodontal ligament
("PL"), oral epithelial ("OE"), odontoblast-like and osteoblast
cell lines have been successfully isolated from Immortomouse by
others and provide convenient model systems for the studies in
dental tissue engineering.
[0074] Prior to seeding the nanofibers with the cells, squares were
cut from the calcium phosphate nanofiber mat, and sterilized by
ultraviolet irradiation for 1 hour per side in a laminar hood.
Alternatively, the fibers were direct spun onto glass cover slips
rendering cutting unnecessary. The fibers were presoaked with
cell-culture medium in a tissue culture plate before seeding. The
fibers settled to the bottom of the plate and got wet easily. The
morphology of cells was then examined by a variety of microscopy
techniques including direct phase contrast visualization of live
cells. In those instances where visible light could not penetrate
the mineralized substrates, fluorescence-labeling of cells was
employed with a Cell/Tracker.TM. green probe followed by
observation using epi-fluorescence and confocal microscopy
(http://dbkgroup.org/celltracker/). In addition, cells were
observed for their interactions with the substrate materials by
electron microscopy. Briefly, cells on the surfaces were fixed with
2% glutaraldehyde in PBS for 2 hours at room temperature. After
fixation, the samples were rinsed with phosphate buffered saline
("PBS") before dehydration with a series of graded ethanol
solutions (10,25, 50, 70, 95 and 100%). The cells were dried with a
critical point dryer. The resulting samples were sputter coated
withl Au--Pd and examined with SEM.
[0075] The viability of cells on the nanofiber scaffolds were
observed using LIVE/DEAD viability/cytotoxicity assay (Molecular
Probes L-3224) or Cell/Tracker.TM. green probe (Molecular Probes
(2-2925)
(http://probes.invitrogen.com/servlets/publications?id-444). Cells
were incubated on the nanofibers for at least 24 hours. Living
cells were labeled with fluorescence and examined using a
microscope. The viability of these cells was compared to those
incubated on tissue culture dishes. Cells were collected from
surfaces and resuspended. Cell suspensions were incubated at
37.degree. C. with MTS solution for 3-4 hours in a 96-well plate.
The absorbance at a wavelength of 570 nm was recorded in a
microplate reader.
[0076] The adhesion of cells on the calcium phosphate nanofiber mat
was compared using the percentage of remaining cells per unit area
on the mat after removing loosely attached cells. The nanofiber
scaffold was placed in culture plates. Fluorescence labeled cells
(CellTracker) were added onto the nanofiber scaffolds and incubated
for about 1 hour, depending on cell type. Unbound cells were
removed by gentle rinsing with medium. Cells were imaged and the
number of cells on the scaffolds counted.
[0077] The differentiation of osteoblast cells focuses on the
analysis of the alkaline phosphatase ("ALP") activity, collagen
synthesis and osteopontin and osteocalcin production. ALP activity
was assessed by staining the cells on the nanofibers using a
diazolim coupling reaction (Fast Blue RR Salt and naphthol AS-MX
phosphate alkaline solution, Sigma, www.sigmaaldrich.com).
Qualitative measurement of ALP activity was determined by analyzing
the cell lysate. Cells were released from the surfaces with the
trypsin/EDTA solution and lysed. Cell lysate was then incubated
with p-nitrophenyl phosphate in an appropriate buffer (Sigma), and
the ALP phosphatase activity was evaluated by monitoring the
absorbance at 405 nm corresponding to the production of
p-nitrophenol. Protein content was measured using BCA assay
(Pierce,
http://www.piercenct.com/products/browse.cfm?fldID=020201). The
activity was expressed as n mlol of
p-nitrophenol/min/mg/protein.
[0078] Collagen synthesis was detected using a histologic staining
procedure (Mason's staining, Sigma). For quantitative comparison,
hydroxyproline concentration was used to represent total collagen
production using the method by Woessner (Woessner. Arch. Biochem.
Biophys. 93: 440-447 (1961)). Samples were mixed with concentrated
HCl and incubated at 110.degree. C. for 16 hours. Hydroxyproline
was oxidated with chloramine T solution and the Ehrlich's reagent
(Sigma) added for color reaction. Absorbance was measured at 560 nm
using a plate-reader.
[0079] Osteopontin and osteocalcin production were determined
quantitatively using ELISA kits available from Assay Design and
Zymed (www.invitrogen.com). The standard protocol provided by the
manufacturer was followed.
[0080] Phenotypical expression of the cell lines isolated from the
Immortomouse on each Ca--P nanofiber scaffold was analyzed at the
mRNA level. Poly(A)-RNA was extracted using Microfastrack method
(Invitrogen, www.invitrogen.com). The expression of mRNA for
specific protein markers of the cell was determined.
[0081] Numerous references, including patents and various
publications, are cited and discussed in the description of this
invention. The citation and discussion of such references is
provided merely to clarify the description of the present invention
and is not an admission that any reference is prior art to the
invention described herein. All references cited and discussed in
this specification are incorporated herein by reference in their
entirety. Variations, modifications and other implementations of
what is described herein will occur to those of ordinary skill m
the art without departing from the spirit and scope of the
invention. While certain embodiments of the present invention have
been shown and described, it will be obvious to those skilled in
the art that changes and modifications may be made without
departing from the spirit and scope of the invention. The matter
set forth in the foregoing description and accompanying drawings is
offered by way of illustration only and not as a limitation. The
actual scope of the invention is intended to be defined in the
following claims.
* * * * *
References