U.S. patent application number 15/321542 was filed with the patent office on 2017-07-27 for zwitterionic hydrogels for delivery of biomolecules.
The applicant listed for this patent is University of Massachusetts. Invention is credited to Pingsheng Liu, Jie Song.
Application Number | 20170209625 15/321542 |
Document ID | / |
Family ID | 55065074 |
Filed Date | 2017-07-27 |
United States Patent
Application |
20170209625 |
Kind Code |
A1 |
Song; Jie ; et al. |
July 27, 2017 |
ZWITTERIONIC HYDROGELS FOR DELIVERY OF BIOMOLECULES
Abstract
The invention provides a novel approach in which zwitterionic
networks are used to sequester and deliver ionic biomolecules, such
as proteins, without compromising their native conformation and
bioactivity. Zwitterionic networks are designed to effectively
retain and deliver ionic or polar biomolecules for guided tissue
regeneration. The invention represents a conceptual advance and
enables a novel strategy for the utilization of zwitterionic motifs
as therapeutics delivery vehicles and tissue engineering
scaffolds.
Inventors: |
Song; Jie; (Shrewsbury,
MA) ; Liu; Pingsheng; (Worcester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Massachusetts |
Boston |
MA |
US |
|
|
Family ID: |
55065074 |
Appl. No.: |
15/321542 |
Filed: |
July 6, 2015 |
PCT Filed: |
July 6, 2015 |
PCT NO: |
PCT/US15/39227 |
371 Date: |
December 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62022187 |
Jul 8, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/18 20130101;
A61L 2300/414 20130101; A61L 27/52 20130101; A61L 27/50 20130101;
A61K 38/30 20130101; A61L 27/58 20130101; A61K 38/1875 20130101;
A61K 38/18 20130101; A61L 27/14 20130101; A61L 2430/02 20130101;
A61L 27/54 20130101 |
International
Class: |
A61L 27/58 20060101
A61L027/58; A61K 38/18 20060101 A61K038/18; A61L 27/18 20060101
A61L027/18; A61L 27/54 20060101 A61L027/54; A61L 27/52 20060101
A61L027/52 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with Government support under grant
no. AR055615 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A composite material comprising a polymer network and a
biologically active compound, wherein the 3-dimensioanl polymer
network comprises a zwitterionic moiety.
2. The composite material of claim 1, wherein the biologically
active compound is a biomacromolecule.
3. The composite material of claim 1, wherein the biomacromolecule
is an ionic or polar protein or peptide.
4. The composite material of claim 3, wherein the biomacromolecule
is a therapeutic osteogenic protein or anabolic agent for bone
formation.
5. The composite material of claim 4, wherein the therapeutic
osteogenic protein or anabolic agent or angiogenic factor is
selected from BMPs, TGF-beta, EGF, FGF, IGF-1 and VEGF.
6. The composite material of claim 5, wherein the therapeutic
osteogenic protein is present in the polymer network at a loading
from about 1 ng to about 20,000 ng.
1. usly Presented) The composite material of claim 1, wherein the
polymer network is a 3-dimensioanl crosslinked polymer network.
8. The composite material of claim 7, wherein the 3-dimensional
polymer network is a crosslinked hydrogel of polymethacrylate,
polyacrylate or polymethacrylamide, polyacrylamide.
9. The composite material of claim 7, wherein the 3-dimensional
polymer network is crosslinked with a crosslinker selected from
poly(ethylene glycol) dimethacrylate, poly(ethylene glycol)
diacrylate, ethylene glycol diacrylate and ethylene glycol
dimethacrylate, or derivatives thereof.
10. The composite material of claim 1, wherein the zwitterionic
moiety comprises one or more selected from sulfobetaine,
phosphorylcholine and carboxybetaine.
11. The composite material of claim 1, wherein the zwitterionic
moiety is present in the polymer network as pendant groups to a
polymeric backbone.
12. The composite material of claim 1, wherein the zwitterionic
moiety is present in the polymer network at a density from about
0.05 mol % to about 10 mol %.
13. The composite material of claim 7, wherein the polymer network
is crosslinked with a crosslinking density from about 1 mol % to
about 100 mol %.
14. The composite material of claim 1, wherein the composite
material is biodegradable.
15. An implant comprising a composite material according to claim
1.
16. An implant comprising a composite material characterized by a
3-dimensional crosslinked polymer network sequestered therein one
or more biologically active compounds, wherein the polymer
comprises a zwitterionic moiety.
17-21. (canceled)
22. The implant of claim 16, wherein the polymer network is a
3-dimensioanl crosslinked polymer network.
23. The implant of claim 22, wherein the 3-dimensional polymer
network is a crosslinked hydrogel of polymethacrylate,
polyacrylate, polymethacrylamide or polyacrylamide.
24. The implant of claim 22, wherein the 3-dimensional polymer
network is crosslinked with a crosslinker selected from
poly(ethylene glycol) dimethacrylate, poly(ethylene glycol)
diacrylate, ethylene glycol diacrylate and ethylene glycol
dimethacrylate, or derivatives thereof.
25-39. (canceled)
40. A method for making a composite material useful for tissue
engineering, comprising: crosslinking, in the presence of a
biologically active compound, a polymer comprising a zwitterionic
moiety to form a 3-dimensioanl crosslinked polymer network with the
biologically active compound encapsulated therein.
41-53. (canceled)
Description
PRIORITY CLAIMS AND RELATED PATENT APPLICATIONS
[0001] This application claims the benefit of priority from U.S.
Provisional Application Ser. No. 62/022,187, filed on Jul. 8, 2014,
the entire content of which is incorporated herein by reference in
its entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] The invention generally relates to biomaterials, tissue
engineering and delivery of biomolecules. More particularly, the
invention relates to zwitterionic polymers and methods of their
applications in delivery of biomolecules and tissue
engineering.
BACKGROUND OF THE INVENTION
[0004] Zwitterions, including phosphobetaine, sulfobetaine, and
carboxybetaine, are well-known for their anti-biofouling properties
as widely demonstrated on 2-dimensional (2D) surfaces. The unique
zwitterionic structures, simultaneously possessing cationic and
anionic residues yet overall electronic neutral, exhibit strong
affinity for water, thereby giving rise to super hydrophilic
surfaces suppressing the hydrophobic interactions known to denature
proteins. (Lowe, et al. 2002 Chemical reviews 102:4177-89; Seo, et
al. 2008 Biomaterials 29:1367-76; Krishnan, et al. 2008 J Mater
Chem 18:3405-13; Kane, et al. 2003 Langmuir 19:2388-91.)
[0005] Zwitterionic motifs have also been shown to mimic the action
of protein stabilizing ions in stabilizing/maintaining the native
conformation of proteins and inhibiting non-specific protein
adsorption, which is known to set off undesired cascades of surface
events (e.g., thrombosis, immune response). Accordingly, they have
been largely exploited for constructing anti-fouling
surfaces/interfaces to inhibit protein, bacterial and cellular
adhesions, and as bioinert implants for reducing scar tissue
formation. (Nakaya, et al. 1999 Prog Polym Sci 24:143-81; Zhang, et
al. 2003 Biomaterials 24:4223-31; Jiang, et al. 2010 Adv Mater
22:920-32; Smith, et al. 2012 Sci Transl Med 4, 153; Ishihara, et
al. 1998 J Biomed Mater Res 39:323-30; Yuan, et al. 2003 Colloid
Surface B 29:247-56; Franz H. Zur Lehre von der Wirkung der Salze.
Archiv fur Experimentelle Pathologie and Pharmakologie 1888;25;
Han, et al. 2007 Sci China Ser B 50:660-4; Keefe, et al. 2012 Nat
Chem 4:60-4; Zhang, et al. 2013 Nat Biotechnol 31:553-6; Harris J
M. Poly(ethyleneglycol) chemistry: biotechnical and biomedical
applications. New York: Plenum Press; 1992; Horbett, et al. 1995
Proteins at Interfaces II: Fundamentals and Applications
Washington, DC: Am. Chem. Soc.)
[0006] Recently, the use of zwitterionic sublfobetaine hydrogel to
facilitate templated biomineralization was reported, which
capitalizes on the ability of the zwitterionic motifs to
effectively recruit/nucleate oppositely charged mineralization
precursor ions (e.g., Ca.sup.2+, PO.sub.4.sup.3-) across the 3D
hydrogel network. (Liu, et al. 2013 Biomaterials 34:2442-54.)
[0007] There is little report, however, on whether 3-dimensionally
presented zwitterions can effectively sequester ionic biomolecules.
Such a property, if intrinsically exists, could fundamentally
change the current perception of zwitterionic materials as being
primarily anti-biofouling and significantly broaden its potential
use in biomedical applications. It is strongly desired that novel
methods and compositions are uncovered and developed that greatly
expand the utility of zwitterionic materials in the bioengineering
and therapeutics areas.
SUMMARY OF THE INVENTION
[0008] The invention provides a novel approach in which
zwitterionic materials are utilized to retain and deliver ionic
biomolecules, such as proteins, for guided tissue regeneration. The
invention uncovers and takes advantage of the ability of
zwitterionic networks to sequester ionic biomacromolecules without
compromising their native conformation and bioactivity, which
challenges the conventional narrow perception and categorization of
zwitterionic materials as low-fouling and bioinert. The invention
demonstrates that zwitterionic networks are versatile vehicles
useful in engineering controlled bioactive microenvironment for
biomedical applications.
[0009] The invention represents a conceptual advance and enables a
novel strategy for the utilization of zwitterionic motifs as
therapeutics delivery vehicles and tissue engineering scaffolds.
The invention distinguishes zwitterionic materials from the current
benchmark biocompatible and anti-fouling material poly(ethylene
glycol) (PEG) that is widely used in the biomaterials field. The
ability of the zwitterionic hydrogel to promote the functional bone
healing with an exceptionally low dose of therapeutic proteins, as
demonstrated herein, can significantly reduce the cost as well as
improve the safety associated with the protein therapeutics.
[0010] In one aspect, the invention generally relates to a
composite material comprising a polymer network and a biologically
active compound, wherein the 3-dimensioanl polymer network
comprises a zwitterionic moiety.
[0011] In another aspect, the invention generally relates to an
implant comprising a composite material characterized by a
3-dimensional crosslinked polymer network sequestered therein one
or more biologically active compounds, wherein the polymer
comprises a zwitterionic moiety.
[0012] In yet another aspect, the invention generally relates to an
implant comprising a 3-dimensional scaffold comprising a
3-dimensioanl polymer network, wherein the polymer network
comprises a zwitterionic moiety, adapted to sustained in vivo
delivery of one or more biologically active compounds.
[0013] In yet another aspect, the invention generally relates to an
implant comprising a n implant comprising a composite material
characterized by a 3-dimensional crosslinked polymer network
comprising a zwitterionic moiety.
[0014] In yet another aspect, the invention generally relates to a
method for making a composite material useful for tissue
engineering. The method includes crosslinking, in the presence of a
biologically active compound, a polymer comprising a zwitterionic
moiety to form a 3-dimensioanl crosslinked polymer network with the
biologically active compound encapsulated therein.
[0015] In yet another aspect, the invention generally relates to a
method for making a composite material useful for tissue
engineering. The method includes: crosslinking a polymer comprising
a zwitterionic moiety to form a 3-dimensioanl crosslinked polymer
network; and contacting the crosslinked polymer network with a
solution of a biologically active compound under conditions such
that the biologically active compound is sequestered in the
crosslinked polymer network.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. Schematic illustrations of (a), the well-established
anti-biofouling property of 2D zwitterionic surfaces vs (b),
hypothesized protein-sequestering property of 3D zwitterionic
networks.
[0017] FIG. 2. 3D zwitterionic hydrogel networks efficiently
sequestered rhBMP-2 and enabled its sustained in vitro release.
(a), Sequestration of rhBMP-2 by zwitterionic PSBMA vs non-ionic
PEGMA control as a function of crosslinker content (n=3, 0.33, 1.33
or 5.33 mol % relative to monomers) after 2-h incubation in PBS. A
300-ng rhBMP-2 initial loading dose was applied to all hydrogels
and the sequestered protein content was determined after 2-h
incubation in PBS. (b), Swelling ratio by weight (S.sub.w) of PSBMA
vs PEGMA hydrogels (n=5) in PBS as a function of crosslinker
content (0.33, 1.33 or 5.33 mol % relative to monomers). (c),
Swelling ratio by weight (S.sub.w) of PSBMA vs PEGMA hydrogels
(1.33 mol % crosslinker content; n=5) in water and in PBS. (d),
Cumulative release of the loaded 300-ng rhBMP-2 from three types of
zwitterionic hydrogels with identical crosslinker amount of 1.33
mol % (n=3). (e), Osteogenic trans-differentiation of C2Cl2 cells
induced by the rhBMP-2 sustained-released (between day 7 to day 9)
from PSBMA vs PEGMA hydrogels as shown by the expression of
osteogenic marker ALP (red stains). C2C12 culture directly
supplemented with 300-ng rhBMP-2 without any hydrogel carrier
served as a positive control.*p<0.05 (two-way ANOVA).
[0018] FIG. 3. High-efficient in vivo local delivery of rhBMP-2 by
PSBMA hydrogel implant as examined by the 5-mm rat femoral
segmental defect model. (a), A PSBMA hydrogel implant (5 mm.times.3
mm.times.3 mm) with/without rhBMP-2 press-fit within the femoral
segmental defect stabilized by a radiolucent polyetheretherketone
(PEEK) plate fixator. (b), Reconstructed .mu.-CT 3D images & 2D
bone mineral density color mapping of the center longitudinal slice
of the defect treated with PSBMA hydrogel grafts with/without
500-ng rhBMP-2 at 4 and 12 weeks post-op. (c), Bone volume &
(d), Bone mineral density of the defects (n=4) treated with PSBMA
hydrogel grafts with/ without 500-ng rhBMP-2 at 4, 8 and 12 weeks
post-op. *p<0.05 (two-way ANOVA) (e), Peak torque of the 12-week
explants treated with PSBMA hydrogel grafts with/without rhBMP-2
(n=3) vs age-matched intact femurs (n=6). *p<0.05 (Student's
T-test). (f), Reconstructed .mu.-CT 3D image & 2D bone mineral
density color mapping of the center transverse slice of the defect
treated with PSBMA hydrogel graft with 500-ng rhBMP-2 at 12 weeks
post-op showing mature bony callus fully encapsulating the rhBMP-2
loaded PSBMA hydrogel scaffold. (g), H&E staining of the
12-week explant showing robust new bone (NB) fully encapsulating
the rhBMP-2 loaded PSBMA scaffold and integrated with adjacent
native cortical bone (CB). BM=bone marrow. Black arrows in the
enlarged image denote hydrogel scaffolds integrated with the
NB.
[0019] FIG. 4. Temporally sequestered rhBMP-2 increased the cell
attachment & ECM deposition on the low-fouling zwitterionic
PSBMA hydrogel implant. (a), Confocal images of in vivo endogenous
cell attachment on the surface of PSBMA explants with/without
rhBMP-2 at day 2 and 7 post-op. Actin was stained by Alexa
phalloidin (red) while nuclei were stained by DAPI (blue). (b),
H&E staining of the ECM deposition on the explants with/without
rhBMP-2 at day 2 and 7 post-op.
[0020] FIG. 5. Mineralization outcomes of zwitterionic pSBMA vs
non-ionic pHEMA hydrogels as examined by SEM and mCT. All hydrogels
were crosslinked by 1.33 mol % of EGDMA relative to monomers. The
hydrogels were placed in an aqueous acidic solution of
hydroxyapaptite (pH=2.5-3.0, 14.7 mg/mL) containing 2-M urea, and
subjected to controlled heating from 37.degree. C. to 95.degree. C.
at 0.2 .degree. C/min. In the absence of ionic motifs, the
mineralization of the non-ionic pHEMA hydrogel occurred exclusively
on the surface. With both positive and negative charged residues
facilitating the penetration of oppositely charged mineralization
precursor ions (e.g. Ca.sup.2+, PO.sub.4.sup.3-) across the
hydrogel, the zwitterionic pSBMA templated extensive mineralization
throughout the 3D hydrogel.
[0021] FIG. 6. Schematic illustration of the preparation of
hydrogels from PEG and zwitterionic methacrylate monomers, and the
loading of rhBMP-2 solutions on hydrogels though the
de-swelling/swelling process. All hydrogels were prepared with
identical crosslinker content of 1.33 mol % relative to
monomers.
[0022] FIG. 7. Cumulative in vitro release of rhBMP-2 from (a)
zwitterionic PSBMA and (b) non-ionic PEGMA hydrogels (n=3) as a
function of PEGDMA crosslinker content in PBS (pH 7.4) as
determined by the BMP-2 Quantikine kit (R&D Systems). Initial
rhBMP-2 loading dose: 300 ng/hydrogel (cylindrical) specimen.
[0023] FIG. 8. Free water fraction (R.sub.f) of PSBMA vs PEGMA
hydrogels (n=3) equilibrated in PBS as determined by DSC. The
difference between the two groups is significant (p<0.05,
Student's T-test).
[0024] FIG. 9. Radiographic monitoring over time of the bony callus
formation over the 5-mm rat femoral segmental defects treated with
PSBMA hydrogel grafts with/without 500-ng rhBMP-2.
[0025] FIG. 10. Reconstructed .mu.-CT 3D images, longitudinal
cross-section views, and longitudinal 2D bone mineral density color
mapping of the 5-mm femoral segmental defect treated with PSBMA
hydrogel grafts with 500-ng rhBMP-2 over time.
[0026] FIG. 11. Reconstructed .mu.-CT 3D images, longitudinal
cross-section views, and longitudinal 2D bone mineral density color
mapping of the 5-mm femoral segmental defect treated with PSBMA
hydrogel grafts alone over time.
[0027] FIG. 12. Reconstructed .mu.-CT 3D images of all 5-mm femoral
segmental defects treated with PSBMA hydrogel implants with/without
the loading of 500-ng rhBMP-2 at 12 weeks post-op.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The invention provides a novel approach in which
zwitterionic networks are used to sequester and deliver ionic
biomolecules, such as proteins, without compromising their native
conformation and bioactivity. Zwitterionic networks are designed to
effectively retain and deliver ionic or polar biomolecules for
guided tissue regeneration. The invention represents a conceptual
advance and enables a novel strategy for the utilization of
zwitterionic motifs as therapeutics delivery vehicles and tissue
engineering scaffolds.
[0029] In contrast to the conventional narrow perception and
categorization of zwitterionic materials as low-fouling and
bioinert, the invention greatly expands the utilities of
zwitterionic materials in the bioengineering and therapeutic areas.
Zwitterionic networks are demonstrated as effective and versatile
vehicles for engineering controlled bioactive microenvironment for
biomedical applications. The invention distinguishes zwitterionic
materials from the current benchmark biocompatible and anti-fouling
material poly(ethylene glycol) (PEG) that is widely used in the
biomaterials field. As demonstrated herein, the ability of the
zwitterionic hydrogel to promote the functional bone healing with
an exceptionally low dose of therapeutic proteins can significantly
reduce the cost and improve the safety associated with the protein
therapeutics.
[0030] For example, as disclosed herein, 3-dimensionally (3D)
presented zwitterionic motifs (e.g., in crosslinked hydrogels),
effectively sequestered osteogenic bone morphogenetic protein-2
(rhBMP-2). The ionic interactions between rhBMP-2 and the 3D
zwitterionic network enabled dynamic sequestering and sustained
release of the protein with preserved bioactivity. The zwitterionic
hydrogel allowed high-efficiency in vivo local delivery of rhBMP-2,
which can template the functional healing of critical-size femoral
segmental defects in rats with rhBMP-2 at a loading dose
substantially lower than those required for current natural or
synthetic polymeric carriers. The sequestered rhBMP-2 can be
sustainedly released well over a week with well-preserved
bioactivity, driven by the dynamic ionic interactions of rhBMP-2
with the 3-dimensionally presented zwitterionic motifs rather than
by scaffold biodegradations.
[0031] Such sequestration and high-efficiency delivery of rhBMP-2
allowed robust repair of critical-size rat femoral segmental
defects templated by the zwitterionic hydrogel implant at an
exceptionally low loading dose of 500-ng rhBMP-2.
[0032] Zwitterions (e.g., phosphobetaine, sulfobetaine, and
carboxybetaine) are well known for their anti-biofouling properties
as widely demonstrated on 2-dimensional (2D) surfaces (FIG. 1a).
The unique zwitterionic structures, simultaneously possessing
cationic and anionic residues yet overall electronic neutral,
exhibit strong affinity for water, thereby giving rise to super
hydrophilic surfaces suppressing the hydrophobic interactions known
to denature proteins. Zwitterionic sublfobetaine hydrogel have been
reported to facilitate templated biomineralization was reported,
which capitalizes on the ability of the zwitterionic motifs to
effectively recruit/nucleate oppositely charged mineralization
precursor ions (e.g., Ca.sup.2+, PO.sub.4.sup.3-) across the 3D
hydrogel network. (Liu, et al. 2013 Biomaterials 34:2442-54.)
Unlike non-ionic hydrogel that was only able to template the
mineralization on the surface, the zwitterionic hydrogel enabled
extensive mineralization throughout the 3D network, supporting the
critical role of zwitterionic motifs in recruiting precursor ions
(FIG. 5). Prior to the disclosure herein, there has been no report
on whether 3D zwitterionic motifs can effectively sequester ionic
biomacromolecules, such as protein, their effective retention and
sustained release (FIG. 1b).
[0033] Effective sequestration of proteins by 3D zwitterionic
hydrogels
[0034] As examples, simple crosslinked polymethacrylate hydrogels
bearing zwitterionic side chains were prepared. The in vitro
sequestration/release profile of osteogenic human recombinant bone
morphogenetic protein-2 (rhBMP-2) from the zwitterionic hydrogels
was investigated and compared with that of the non-ionic
low-fouling poly(ethylene glycol) hydrogel control. The efficacy of
a zwitterionic sulfobetaine hydrogel in delivering rhBMP-2 in vivo
to promote the functional healing of critical-size (5-mm) femoral
segmental defects in rats and endogenous cell attachment on the
otherwise low-fouling implant was investigated.
[0035] The zwitterionic PSBMA hydrogels were prepared by
photo-crosslinking sulfobetaine methacrylate (SBMA) with varying
contents of crosslinker PEGDMA. And a poly(ethylene glycol)
methacrylate (PEGMA) hydrogels bearing non-ionic poly(ethylene
glycol) (PEG), another well-established anti-biofouling motif, were
prepared at the identical crosslinker contents as controls (FIG. 6
& Table 1). To examine the efficiency of the hydrogels for
sequestering therapeutic proteins, 300 ng of rhBMP-2 (in 10 .mu.L
PBS solution) was loaded on each partially dried hydrogel and
allowed to equilibrate at 37.degree. C. for 1 h to ensure complete
absorption of the aqueous solution (FIG. 6).
TABLE-US-00001 TABLE 1 Formulations of the photo-crosslinked
hydrogels with identical crosslinker content VA-086 Monomer
Crosslinker stock amount amount.sup.a solution.sup.b PBS Monomer
(mmol) (.mu.L) (.mu.L) (.mu.L) SBMA 2 17.9 100 1882.1 PEGMA 2 17.9
100 1882.1 MPC 2 17.9 100 1882.1 CBMA 2 17.9 100 1882.1
.sup.aPEGDMA (M.sub.n = 750). .sup.b2% (w/v) VA-086 in PBS.
[0036] Although zwitterionic sulfobetaine and PEG surfaces are both
known for resisting non-specific protein absorptions, the
respective 3D networks exhibited significant differences in
sequestering rhBMP-2 even with a similar swelling ratio at the
identical crosslinker content of 5.33 mol % (relative to monomer;
FIGS. 2a & 2b). The non-ionic PEGMA hydrogel could not
effectively sequester rhBMP-2, with only about 10% of the initially
loaded rhBMP-2 retained on the hydrogel after 2-h incubation in PBS
(FIG. 2a). This observation is consistent with previous findings
that PEG hydrogels lack affinity for ionic proteins. (Zhu 2010
Biomaterials 31:4639-56; Place, et al. 2009 Nature Materials
8:457-70.) By contrast, about 60% of the initially loaded rhBMP-2
was sequestered by the zwitterionic PSBMA network of the same
crosslinker content (5.33 mol %) after 2-h incubation (FIG. 2a).
Given the similar swelling ratio, thus similar diffusibility of
solutes across the 3D network, the different efficiencies of
sequestering ionic proteins observed with the two identically
crosslinked networks was likely due to the different ionic states
of their side chains. (Hoffman 2002 Advanced drug delivery reviews
54:3-12.)
[0037] By reducing the degree of chemical crosslinking by up to
16-fold, it was shown that the zwitterionic PSBMA network swelled
significantly in PBS by up to 10-fold while no significant
crosslinker content-dependent changes in swelling ratio in PBS was
observed with the non-ionic PEGMA network (FIG. 2b). This further
supported that the different ionic states of side chains presented
in the two 3D networks (zwitterionic vs non-ionic) can translate
into significant differences in their physical properties in ionic
environment, including different swelling behavior and efficiencies
in sequestering/releasing ionic protein (FIGS. 2a and 7).
[0038] Furthermore, unlike the non-ionic PEGMA network that was
insensitive to the presence of salts (no significant difference in
swelling ratios in water vs in PBS, pH 7.4, FIG. 2c), the
zwitterionic PSBMA network expanded almost 400% more in PBS than in
water (FIG. 2b). Such an antipolyelectrolyte swelling behavior can
be attributed to the disruption of the intermolecular salt bridges
formed between the anionic sulfonate and cationic ammonium residues
by ionic solutes. (Poynton, et al. 2002 Spine 27:S40-S8.) Combined
with the higher free water fractions in the equilibrated
zwitterionic PSBMA hydrogel (85% in PSBMA vs 69% in PEGMA, FIG. 8),
this observation further supports that the ionic-sensitive nature
of the zwitterionic network is beneficial to the diffusion of ionic
solutes in general across the 3D network.
[0039] Taken together, these data validate that ionic interactions
play an indispensable role in effectively sequestering rhBMP-2 by
the zwitterionic PSBMA network. Similar rhBMP-2 retention profiles
were also observed with the 3D zwitterionic networks bearing
phosphobetaine (PMPC) and carboxybetaine (PCBMA) motifs (FIG. 2d),
supporting effective protein retention as a novel yet generalizable
feature for 3D zwitterionic matrices.
Sustained Release of Bioactive Proteins from Zwitterionic
Hydrogels
[0040] Monitoring of the rhBMP-2 release from the hydrogels within
the first 24 h of incubation in PBS by ELISA revealed .about.30%
release of the initially loaded protein in the first 2 h, followed
by a 3% of slower release in the next 22 h (FIG. 2d), leaving
>65% sequestered by the zwitterionic PSBMA (1.33 mol %
crosslinker content) by 24 h.
[0041] To examine whether the rhBMP-2 sequested by the PSBMA
hydrogel could be continually released with retained bioactivity
over a much longer period of time, an established culture model of
BMP-2-induced osteogenic trans-differentiation of murine myoblast
C2Cl2 cells was used. (Katagiri, et al. 1994 J Cell Biol
127:1755-66; Liu, et al. 2011 Acta Biomaterialia 7:3488-95.) This
model was chosen over BMP-2-induced osteogenesis of mesenchymal
stem cells (MSCs) due to the complete lack of expression of
osteogneic markers by C2Cl2 cells prior to BMP-2 induction (thus
much cleaner background than MSCs). It was shown that when the
rhBMP-2-bearing PSBMA was placed in murine myoblast C2Cl2 cutlure
after a 6-day pre-incubation in PBS, the further sustainaedly
released rhBMP-2 (from day 7 to day 9) from the PSBMA hydrogel was
able to induce robust osteogenic trans-differentiation of C2Cl2
cells into alkaline phosphatase (ALP)-expressing osteoblasts (FIG.
2e). The intense ALP staining, comparable to that observed with the
positive control culture (FIG. 2e) where 300-ng rhBMP-2 was
directly supplemented without any carrier, suggest that the
bioactivity of the sequestered and subsequently released rhBMP-2
was well preserved for at least 9 days.
[0042] This result is in stark contrast to the minimal ALP stains
detected from the C2Cl2 culture supplemented with the PEGMA
hydrogel subjected to identical BMP-2 loading and PBS
pre-incubation treatment, consistent with the much poorer initial
sequestration of rhBMP-2 by the non-ionic PEGMA hydrogel. It is
worth noting that the circulation half-life of rhBMP-2 and most
other growth factors, when in free form, tends to be very limited
(e.g., 7-16 min for rhBMP-2). (Poynton, et al. 2002 Spine
27:S40-S8.) Here, well-preserved bioactivity of the rhBMP-2
sequestered by the PSBMA hydrogel was demonstrated well over a
week. This may be attributed to the superhydrophilic structrual
water surrounding zwitterioic residues that prevent protein
denaturing and the Hofmeister ions-like effect of the zwitterions
for stabilizing native protein conformations. (Kane, et al. 2003
Langmuir 19:2388-91; Franz H. Zur Lehre von der Wirkung der Salze.
Archiv fur Experimentelle Pathologie and Pharmakologie 1888;25;
Keefe, et al. 2012 Nat Chem 4:60-4.) Overall, these observations
support the zwitterionic PSBMA hydrogel as an effective carrier for
the high-efficiency sequestration and sustained long-term release
of theapeutic proteins such as rhBMP-2.
Treating Bone Defects by High-Efficiency In Vivo Delivery of
Proteins
[0043] To test the in vivo efficacy of the PSBMA hydrogel as a
synthetic implant with rhBMP-2 delivery capability, the repair of
5-mm rat femoral segmental defect, an established critical-size
non-union model, templated by the PSBMA implant with or without
pre-loaded rhBMP-2 was evaluated (FIG. 3a). (Filion, et al. 2011
Tissue Eng Pt A 17:503-11; Uhrig, et al. 2013 Bone 55:410-7.)
[0044] Current clinical use of rhBMP-2, delivered via absorbable
collagen sponge carrier (INFUSE.RTM.), to stimulate spine fusion or
tibial fracture repair require exceedingly high loading doses
comparable to .about.1.5 mg per milliliter volume of defect (1500
ng/mm.sup.3). Such a supra-physiological dosages and their burst
release from the sub-optimal collagen carrier have resulted in
significant systemic and local adverse effects. Loading doses
ranging from 2 to 50-.mu.g rhBMP-2/scaffold (.about.250 to 6,250
ng/mm-defect) have been typically used to achieve adequate repair
of critical-size long bone or trabecular bone defects in rats with
either natural or synthetic polymeric carriers. Table 2 lists
representative reported rhBMP-2 loading doses on various natural or
synthetic polymeric scaffolds used for achieving adequate healing
of critical-size bone defects in rats. Literatures reporting
synergistic loading of rhBMP-2 along with other growth
factors/therapeutics are not included.
TABLE-US-00002 TABLE 2 Representative literature rhBMP-2 loading
doses on various natural or synthetic polymeric scaffolds rhBMP-2
loading dose Scaffold Scaffold .mu.g/ .mu.g/mm- .mu.g/ type
materials Defect model scaffold defect mm.sup.3 Natural Collagen
1.5 (INFUSE .RTM.).sup.a Gelatin 6-mm segmental, 3 0.5 ulna
Alginate 8-mm segmental, 2 0.25 femur 5 0.63 Keratose 8-mm
segmental, 50 6.25 femur Silk 5-mm segmental, 2.5 0.5 femur
Hyaluronic 5-mm cranium 5 acid Synthetic PPF/TCP 5-mm segmental, 10
2 femur PLGA & PPF 5-mm segmental, 6.5 1.3 femur PLA-DX-PEG
4-mm, ilia 10 PEG-RGD 8-mm, cranium 5 PSBMA.sup.b 5-mm segmental,
0.5 0.1 0.01 femur .sup.acommercial rhBMP-2 delivery scaffolds
approved by FDA. .sup.bzwitterionic PSBMA hydrogel scaffold used in
the current study. .sup.cReferences: Mckay, et al. 2007 Int'l
orthopaedics 31: 729-34; Carragee, et al. 2011 Spine J 11: 471-91;
Ratanavaraporn, et al. 2011 Biomaterials 32: 2797-811; Kolambkar,
et al. 2011 Bone 49: 485-92; de Guzman, et al. 2013 Biomaterials
34: 1644-56; Kirker-Head, et al. 2007 Bone 41: 247-55; Patterson,
et al. 2010 Biomaterials 31: 6772-81; Kempen, et al. 2009
Biomaterials 30: 2816-25; Lutolf, et al. 2003 Nat Biotechnol 21:
513-8; Chu, et al. 2007 Biomaterials 28: 459-67; et al. 2001 Nat
Biotechnol 19: 332-5.
[0045] Loading doses of rhBMP-2 less than 2 .mu.g (without
synergistic delivery of other growth factor therapeutics) often
resulted in inadequate/inconsistent repair outcomes. (Schmoekel, et
al. 2005 Biotech. & bioeng. 89:253-62.) Here, in contrast, a
significantly lower loading dose of 500-ng rhBMP-2 was applied to
the PSBMA scaffold (equivalent to .about.11 ng/mm.sup.3 or 100
ng/mm-defect) press-fit into the 5-mm rat femoral segmental defect.
It is belived that consistent functional healing of critical rat
long bone defect with such a low loading dose of rhBMP-2 alone has
never been reported before.
[0046] At 2 weeks, mineralized healing callus emerged around the
defects implanted with PSBMA with rhBMP-2 (FIG. 9). Strikingly, the
bony callus started to bridge over the defect by as early as 4
weeks (FIG. 3b, FIG. 10), and byl2 weeks, mature and uniform bony
callus characterized with recanalization and high bone mineral
density (FIGS. 3b, 3d, 3f, FIG. 10) fully encapsulated the defect,
leading to substantial restoration (.about.40% compared to intact
age matched femur control) of the torsional rigidity of the defect
(FIG. 3e).
[0047] Continued remodeling of the new bone is expected to further
increase the torsional rigidity over time. In the absence of
rhBMP-2, the PSBMA also led to the early onset (FIG. 9) and steady
growth of bony callus over the course of 12 weeks as characterized
with increasing bone volume (FIG. 3c) and bone mineral density
(FIG. 3d). However, in the absence of rhBMP-2, the calcified callus
failed to bridge over the entire defects by 12 weeks (FIGS. 3b, 9,
11 & 12) to restore the biomechanical integrity of the defect
(FIG. 3e). Of note, although fairly high bone volumes were detected
at the regions of interest (ROI) in both treatment groups by 12
weeks (FIG. 3c, no statistically significant difference), the
rhBMP-2 treated group consistently guided uniform bony callus
formation across the full length of the defect whereas the new bone
formation templated by the no-BMP-2 control group was primarily
localized around the graft-cortical bone junctions (FIG. 12).
[0048] Transverse cross-sectional view of the repaired defect (FIG.
3f) and H&E staining of the explant at 12-week post-op (FIG.
3g) revealed that the bony callus formation was tightly templated
by and integrated with the PSBMA hydrogel (note that the
disintegration/shrinkage of hydrogel scaffold trapped within the
bony callus was a histology processing artifact as the hydrogel
shrank dramatically upon dehydration).
[0049] These data supported that PSBMA hydrogel implant is a highly
effective carrier for the local delivery of rhBMP-2, which enabled
the functional repair of rat critical-size long bone defect at a
significantly reduced BMP-2 loading dose that is desired from both
safety and cost-effectiveness perspectives.
rhBMP-2 Sequestration Promoting Endogenous Cell Attachment &
ECM Deposition on the Otherwise Low Fouling Surface of Zwitterionic
PSBMA Hydrogel Implant
[0050] The robust early bone healing enabled by PSBMA in the
presence of rhBMP-2 across the entire defect suggests that a
cascade of cellular events required for initiating bone healing
must have occurred in a timely manner along the implant surface,
counterintuitive to the perception that zwitterionic surfaces and
scaffolds tend to reduce protein absorptions/cellular adhesion.
(Smith, et al. 2012 Sci Transl Med 4, 153; Zhang, et al. 2013 Nat
Biotechnol 31:553-6; Bose, et al. 2012 Trends in biotechnology
30:546-54; Liu, et al. 2009 Biomacromol. 10:2809-16.)
[0051] Also investigated was the early stage in vivo cell
attachment during the guided bone healing with and without the
loading of rhBMP-2. The results showed that the retention of
rhBMP-2 by the PSBMA hydrogel implants shifted the microenvironment
of the zwitterionic scaffolds from low-fouling to cell adhesive. As
revealed by fluorescent microscopy and H&E staining, only
limited cell attachment was observed on the surface of the PSBMA
hydrogel without rhBMP-2 within the first 2 days post-implantation
with no obvious increases by 7 days (FIG. 4). This is consistent
with the low-fouling nature of zwitterionic surfaces as well as the
recent report that zwitterionic carboxybetaine hydrogels suppressed
fibrous tissue encapsulation in vivo. (Zhang, et al. 2013 Nat
Biotechnol 31:553-6)
[0052] In contrast, substantially more endogenous cells attached to
the surface of the rhBMP-2-bearing PSBMA implant at 2 days
post-implantation (FIG. 4), and these adherent cells continued to
proliferate and led to more effective ECM deposition, and
presumably the initiation of callus formation, at day 7
post-implantation. These observations suggest that the ionic
retention of rhBMP-2 on the 3D zwitterionic scaffold not only
introduced osteoinductivity, but also improved the
osteoconductivity of the otherwise commonly perceived low-fouling
and bioinert scaffold, enabling facile cellular attachment. As many
ECM components such as fibronectin, collagen and laminin have high
affinity for heparin-binding growth factors like BMPs, the
rhBMP-2-bearing scaffold in turn could facilitate the attachment of
these ECM components and subsequent cellular adhesion and more
uniform and robust bony callus formation. (Ruoslahti, et al. 1991
Cell 64:867-9.)
[0053] Thus, in one aspect, the invention generally relates to a
composite material comprising a polymer network and a biologically
active compound, wherein the 3-dimensioanl polymer network
comprises a zwitterionic moiety.
[0054] In certain embodiments, the biologically active compound is
a biomacromolecule. In certain embodiments, the biologically active
compound is a small molecule compound. In certain embodiments, the
biologically active compound is a biomacromolecule such as an ionic
or polar protein or peptide.
[0055] In certain preferred embodiments, the biomacromolecule is a
therapeutic osteogenic protein, an anabolic agent or any angiogenic
factor, for example, selected from BMPs (e.g., rhBMP-2, rhBMP-7,
rhBMP-2/7 heterodimer), TGF-beta, EGF, FGF, IGF-1, and VEGF. In
certain embodiments, the therapeutic osteogenic protein is present
in the polymer network at a loading from about 1 ng to about 20,000
ng (e.g., from about 1 ng to about 20,000 ng, from about 10 ng to
about 20,000 ng, from about 100 ng to about 20,000 ng, from about
1,000 ng to about 20,000 ng, from about 5,000 ng to about 20,000
ng, from about 10,000 ng to about 20,000 ng, from about 1 ng to
about 10,000 ng, from about 1 ng to about 5,000 ng, from about 1 ng
to about 3,000 ng, from about 1 ng to about 1,000 ng, from about 1
ng to about 500 ng, from about 1 ng to about 300 ng, from about 1
ng to about 100 ng) per critical-size femoral segmental defect
(e.g., in rat or scaled to human defect sizes accordingly).
[0056] The polymer network is preferably a 3-dimensioanl
crosslinked polymer network. Any suitable polymer network may be
utilized, for example, a crosslinked hydrogel of polymethacrylate,
polyacrylate, polymethacrylamide or polyacrylamide. Any suitable
crosslinkers may be utilized, for example, selected from
poly(ethylene glycol) dimethacrylate, poly(ethylene glycol)
diacrylate, ethylene glycol diacrylate and ethylene glycol
dimethacrylate, or derivatives thereof (e.g., amides). The polymer
network may be crosslinked to any suitable crosslinking density,
for example, from about 0.05 mol % to about 10 mol % (e.g., from
about 0.05 mol % to about 5 mol %, from about 0.05 mol % to about 3
mol %, from about 0.05 mol % to about 1 mol %, from about 0.05 mol
% to about 0.5 mol %, from about 0.05 mol % to about 0.1 mol %,
from about 0.1 mol % to about 10 mol %, from about 0.5 mol % to
about 10 mol %, from about 1 mol % to about 10 mol %, from about 5
mol % to about 10 mol %).
[0057] Any suitable zwitterionic moieties may be incorporated in
the polymer network, for example, one or more selected from
sulfobetaine, phosphorylcholine and carboxybetaine. The
zwitterionic moieties may be present in a polymer network in the
backbone and/or as pendant groups to a polymeric backbone. The
zwitterionic moieties may be present in the polymer network at any
suituable density, for example, from about 1 mol % to about 100 mol
% (e.g., from about 1 mol % to about 50 mol %, from about 1 mol %
to about 30 mol %, from about 1 mol % to about 30 mol %, from about
1 mol % to about 10 mol %, from about 1 mol % to about 5 mol %,
from about 5 mol % to about 100 mol %, from about 10 mol % to about
100 mol %, from about 30 mol % to about 100 mol %, from about 50
mol % to about 100 mol %).
[0058] In certain embodiments, the composite material is
biodegradable.
[0059] In another aspect, the invention generally relates to an
implant comprising a composite material characterized by a
3-dimensional crosslinked polymer network sequestered therein one
or more biologically active compounds, wherein the polymer
comprises a zwitterionic moiety.
[0060] In yet another aspect, the invention generally relates to an
implant comprising a 3-dimensional scaffold comprising a
3-dimensioanl polymer network, wherein the polymer network
comprises a zwitterionic moiety, adapted to sustained in vivo
delivery of one or more biologically active compounds.
[0061] In yet another aspect, the invention generally relates to an
implant comprising a n implant comprising a composite material
characterized by a 3-dimensional crosslinked polymer network
comprising a zwitterionic moiety.
[0062] In certain preferred embodiments, the implant of the
invention is suitable for treating dental, bone, cartilage, tendon,
ligament or osteochondral damage.
[0063] In yet another aspect, the invention generally relates to a
method for making a composite material useful for tissue
engineering. The method includes crosslinking, in the presence of a
biologically active compound, a polymer comprising a zwitterionic
moiety to form a 3-dimensioanl crosslinked polymer network with the
biologically active compound encapsulated therein.
[0064] In yet another aspect, the invention generally relates to a
method for making a composite material useful for tissue
engineering. The method includes: crosslinking a polymer comprising
a zwitterionic moiety to form a 3-dimensioanl crosslinked polymer
network; and contacting the crosslinked polymer network with a
solution of a biologically active compound under conditions such
that the biologically active compound is sequestered in the
crosslinked polymer network.
Experimental
Preparation of Hydrogels
[0065] Zwitterionic hydrogels
poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium
hydroxide (PSBMA), poly(2-Methacryloyloxyethyl phosphorylcholine)
(PMPC),
poly[3-((2-(methacryloyloxy)ethyl)dimethylammonio)propanoate]
(PCBMA) and nonionic poly(ethylene glycol) methacrylate (PEGMA,
Mn=360) were prepared (Table 1). Monomers SBMA, MPC and PEGMA
(Mn=360) and crosslinker poly (ethylene glycol) dimethacrylate
(PEGDMA, Mn=750) were purchased from Aldrich (St. Louis, Mo.),
while CBMA was synthesized as reported. (Zhang, et al. 2006
Langmuir 22:10072-7.) The radical inhibitors in PEGMA and PEGDMA
were removed by passing through an aluminum oxide column prior to
use. In a typical procedure, 2 mmol respective monomer was combined
with 17.9 .mu.L of PEGDMA, 100 .mu.L of PBS solution of
2,2'-Azobis[2-methyl-N-(2-hydroxyethyl)propionamide] (VA-086, 2%,
w/v), and 1882.1 .mu.L of PBS. The mixture was bath-sonicated, and
sterilized by passing through 0.22-.mu.m polyethersulfone (PES)
membrane filter (Millipore). The resulting solution was transferred
to a custom-made Teflon mold with cylindrical (6 mm in diameter, 50
.mu.L/well), square prism (5 mm.times.5 mm, 50 .mu.L/well) or
rectangle (6.5.times.32.6 mm, 400 .mu.L/well) wells and solidified
under the irradiation of 365-nm light for 10 min in a sterile hood.
The hydrogels were stored in sterile PBS until further uses.
Swelling Ratios of the Hydrogels
[0066] The swelling ratios by weight (Sw) of the hydrogels were
determined in Milli-Q water or in PBS (pH=7.4) at room temperature
according to Equation 1:
S w = W h - W d W h Eq - 1 ##EQU00001##
where W.sub.h and W.sub.d are the weight of the hydrogel in fully
hydrated state in water/PBS and freeze-dried state,
respectively.
Free Water Fraction in the Hydrogels
[0067] The free water fraction in the hydrogels was measured by
differential scanning calorimetry (DSC) on a Q200 Modulated DSC (TA
Instruments). About 15 mg of hydrogel equilibrated either in water
or PBS was placed in an aluminum pan. The pan was then sealed
tightly to prevent water evaporation during the measurement. The
testing was carried out from -40 .degree. C. to 40 .degree. C. at a
heating rate of 2.degree. C./min. The exothermal peak around
0.degree. C., attributed to the melting of the free water[19], was
calculated as .DELTA.H.sub.endo, and the free water fraction
(R.sub.f) within the hydrogel was determined according Equation
2:
R f = .DELTA. H endo .DELTA. H w Eq - 2 ##EQU00002##
where .DELTA.H.sub.w is the heat fusion of pure water (332.2
mJ/mg)[6]. In Vitro Retention and Sustained Release of rhBMP-2
[0068] Recombinant protein rhBMP-2 (R&D Systems, CHO-derived)
was reconstituted according to vendor specifications and diluted
with Ca.sup.2+/Mg.sup.2+-free Dulbecco's phosphate-buffered saline
(DPBS, pH 7.4) to a loading concentration of 30 ng/.mu.L. Hydrogels
retrieved from the sterile stock solution were partially dried in a
sterile cell culture hood (with a gel volume reduction of 50 to 100
mm.sup.3), and then transferred into the wells of ultra-low
attachment 24-well plate (Corning). Reconstituted rhBMP-2 solution
(10 .mu.L, 30 ng/.mu.L) was placed on each hydrogel to achieve a
total loading dose of 300-ng rhBMP-2/hydrogel (cylindrical), and
allowed to be incubated at 37.degree. C. for 1 h (during which
rhBMP-2 solutions were fully absorbed by the hydrogels). The
rhBMP-2loaded hydrogels were then incubated in 1 mL of DPBS at
37.degree. C. for 2, 4, 6, 10, and 24 h. Concentration of the
released rhBMP-2 in the DPBS at various time points were determined
by an enzyme-linked immuno sorbent assay (ELISA) using a rhBMP-2
Quantikine Kit (R&D Systems) and the amount of the rhBMP-2
released form hydrogels were calculated from the standard curve
generated during the same experiment. A sample size of 3 was
applied to each hydrogel group.
Bioactivity of the rhBMP-2 Sequestered on & Released from the
Hydrogels
[0069] The bioactivity of the rhBMP-2 retained on and subsequently
released from the PEGMA and PSBMA hydrogels was evaluated by their
ability to induce osteogenic trans-differentiation of murine
myoblast C2Cl2 cells into osteoblasts. (Liu, et al. 2011 Acta
Biomaterialia 7:3488-95; Filion, et al. 2011 Tissue Engineering
Part A 17:503-11.) C2Cl2 cells were seeded on 24-well cell culture
plate (10,000 cells/cm.sup.2) in 1 mL of Dulbecco's modified eagle
medium (DMEM) supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin, and allowed to attach overnight. The
medium was then replaced with fresh DMEM supplemented with 5% fetal
bovine serum and 1% penicillin/streptomycin, and the rhBMP-2 loaded
hydrogels retrieved from prior incubation in PBS up to 6 days were
placed in the adherent C2Cl2 culture. After 3 days, the hydrogel
was removed and the cells were fixed and stained for alkaline
phosphatase (ALP) using a Sigma Leukocyte Alkaline Phosphatase Kit
according to the vender's protocol. C2Cl2 culture directly
supplemented with 300-ng rhBMP-2 without any hydrogel carrier
served as a positive control.
Animal Surgical Procedures
[0070] All animal procedures were approved by the University of
Massachusetts Medical School Institutional Animal Care and Use
Committee. Briefly, male Charles River SASCO-SD rats (289-300 g)
were sedated and maintained by 2% isoflurane--oxygen throughout the
surgery. The mid-shaft of a femur was exposed by a combination of
sharp and blunt dissections and the periosteum of the exposed femur
was circumferentially removed to emulate a challenging clinical
scenario where this important source of progenitor cells and
signaling molecules is lost. (Filion, et al. 2011 Tissue
Engineering Part A 17:503-11.) A radiolucent, weight bearing
polyetheretherketone (PEEK) internal fixation plate was secured to
the exposed femur with four bicortical screws into predrilled
holes. A 5-mm mid-diaphyseal defect was then created using an
oscillating Hall saw with parallel blades. The defect site was
thoroughly irrigated with saline to remove bone debris and residue
detached periosteum before it was press-fit with a hydrogel graft
with or without 500-ng rhBMP-2 (FIG. 3a, n=4). The muscle and skin
were closed with resorbable sutures and the rats were given
cefazolin (20 mg/kg, once a day) and bupenorphine (0.08 mg/kg, 3
times a day) injections subcutaneously over the next 2 days. Rats
were radiographed biweekly post-op to ensure proper graft
positioning, and subjected to monthly longitudinal microCT
(.mu.-CT) scans (n=4) to quantitatively monitor the mineralized
callus formation until time of sacrifice at 12 weeks post-op. For
end-time point analyses, the femur, with the PEEK plate fixator
intact, was carefully separated from the adjacent hip and knee
joints for either torsion test (n=3) or histological staining. In a
second set of experiments, implants were retrieved at 2 and 7 days
post-op (n=2) for examination of cellular attachment on the surface
of the implant.
Longitudinal .mu.-CT Analysis
[0071] Rats were scanned immediately post-op and every 4 weeks
thereafter on a viva-CT 75 in vivo Micro-CT system (SCANCO Medical
AG) to monitor new bone formation over time. The effective voxel
size of the reconstructed images was 30.times.30.times.30
.mu.m.sup.3. Data were globally thresholded and 3D images of the
5-mm defect, defined as the region of interest (ROI, 167 slices, 30
.mu.m/slice), were reconstructed for quantification of bone volume
(BV, mm.sup.3) and bone mineral density (BMD, mgHA/ccm).
Two-dimensional (2D) mineral density color mapping was generated by
reconstructing the respective AIM file with a colored density
gradient range of 1.5-3.5 (l/mm). An unimplanted PSBMA hydrogel was
scanned to guide proper setting of the threshold (to eliminate
hydrogel background) for all analyses.
Torsion Test
[0072] Explanted femora were torqued to failure as previously
described to assess the degree of the functional restoration of
their biomechanical integrity. (Filion, et al. 2011 Tissue Eng Pt A
17:503-11.) Briefly, explant was potted in stainless steel hexanuts
with poly(methyl methacrylate). The PEEK plate fixators were either
carefully bisected without disturbing the underlying graft/new bone
using a high-speed burr (the PSBMA group) or unscrewed and removed
from the explants (rhBMP-2 treated group) before mounted on the
mini-torsion tester (ADMET Inc.). Each specimen was torqued to
failure at 1.degree./s.
Histology
[0073] The explants were fixed by 10% zinc formalin for 24 h,
decalcified in 18% EDTA at 4.degree. C. for 4 weeks, and embedded
with glycol methacrylate and sectioned. The 3-.mu.m sections were
mounted onto slides for hematoxylin & eosin (H&E)
staining.
Early-Stage In Vivo Cell Attachment on Implant Surfaces
[0074] To visualize the in vivo cell attachment to the hydrogel
scaffolds during the early stage of guided bone regeneration, the
hydrogel implants with/without pre-loaded rhBMP-2 (500 ng/hydrogel)
were retrieved at 2 and 7 days post-op. The explants were fixed in
3.7% formaldehyde/DPBS solution, and the adherent cells were
stained with Alexa Fluor.RTM. 488 phalloidin (for F-actin staining,
red) and DAPI (for nuclei staining, blue) following the vendor's
protocol, respectively, and imaged on a Leica TCS SP2 confocal
microscope. Phalloidin was excited at 495 nm and observed with a
518-nm filter while DAPI was excited at 368 nm and observed with a
461-nm filter.
[0075] In this specification and the appended claims, the singular
forms "a," "an," and "the" include plural reference, unless the
context clearly dictates otherwise.
[0076] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Although any methods and materials
similar or equivalent to those described herein can also be used in
the practice or testing of the present disclosure, the preferred
methods and materials are now described. Methods recited herein may
be carried out in any order that is logically possible, in addition
to a particular order disclosed.
INCORPORATION BY REFERENCE
[0077] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made in this disclosure. All such
documents are hereby incorporated herein by reference in their
entirety for all purposes. Any material, or portion thereof, that
is said to be incorporated by reference herein, but which conflicts
with existing definitions, statements, or other disclosure material
explicitly set forth herein is only incorporated to the extent that
no conflict arises between that incorporated material and the
present disclosure material. In the event of a conflict, the
conflict is to be resolved in favor of the present disclosure as
the preferred disclosure.
EQUIVALENTS
[0078] The representative examples are intended to help illustrate
the invention, and are not intended to, nor should they be
construed to, limit the scope of the invention. Indeed, various
modifications of the invention and many further embodiments
thereof, in addition to those shown and described herein, will
become apparent to those skilled in the art from the full contents
of this document, including the examples and the references to the
scientific and patent literature included herein. The examples
contain important additional information, exemplification and
guidance that can be adapted to the practice of this invention in
its various embodiments and equivalents thereof.
* * * * *