U.S. patent application number 12/757351 was filed with the patent office on 2010-08-05 for polymeric resorbable composites containing an amorphous calcium phosphate polymer ceramic for bone repair and replacement.
This patent application is currently assigned to DREXEL UNIVERSITY. Invention is credited to ARCHEL M.A. AMBROSIO, Cato T. Laurencin, JANMEET S. SAHOTA.
Application Number | 20100197823 12/757351 |
Document ID | / |
Family ID | 23052831 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100197823 |
Kind Code |
A1 |
Laurencin; Cato T. ; et
al. |
August 5, 2010 |
Polymeric Resorbable Composites Containing an Amorphous Calcium
Phosphate Polymer Ceramic for Bone Repair and Replacement
Abstract
A method for making bioresorbable composites of a
non-crystalline calcium phosphate ceramic synthesized within
encapsulating microspheres of bioresorbable polymeric material for
use in bone repair and replacement is provided. The composites
include microspheres and scaffolds produced therefrom.
Inventors: |
Laurencin; Cato T.;
(Earlysville, VA) ; AMBROSIO; ARCHEL M.A.; (SAN
DIEGO, CA) ; SAHOTA; JANMEET S.; (NEW PROVIDENCE,
NJ) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
CIRA CENTRE, 12TH FLOOR, 2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Assignee: |
DREXEL UNIVERSITY
PHILADELPHIA
PA
|
Family ID: |
23052831 |
Appl. No.: |
12/757351 |
Filed: |
April 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10469617 |
Feb 6, 2004 |
7727539 |
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PCT/US02/07854 |
Mar 14, 2002 |
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12757351 |
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60275561 |
Mar 14, 2001 |
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Current U.S.
Class: |
523/115 |
Current CPC
Class: |
A61F 2002/30062
20130101; A61F 2/28 20130101; A61F 2210/0004 20130101; A61F
2002/30968 20130101; Y10S 977/70 20130101; A61L 27/46 20130101;
A61F 2310/00293 20130101; A61L 27/50 20130101 |
Class at
Publication: |
523/115 |
International
Class: |
A61F 2/28 20060101
A61F002/28 |
Goverment Interests
GOVERNMENT INTERESTS
[0002] This work was supported in part by the National Science
Foundation (Grant No. NSG-BES9817872) and the U.S. Government has
certain rights in this invention.
Claims
1. A method for producing a bioresorbable composite comprising: (a)
contacting a basic aqueous calcium salt solution and a basic
aqueous phosphate salt solution in a non-polar immiscible solvent
containing at least one bioresorbable polymeric material at a
temperature; and (b) emulsifying the mixture with sufficient energy
for a time and at a temperature sufficient to provide for the
reaction between the calcium and phosphate salts to form a
non-crystalline or amorphous calcium phosphate composition
encapsulated within encapsulating microspheres of the bioresorbable
polymer.
2. The method of claim 1 wherein the calcium salt is calcium
nitrate, or a hydrate thereof.
3. The method of claim 1 wherein the phosphate salt is ammonium
hydrogen phosphate, or a hydrate thereof.
4. The method of claim 1 wherein the basic calcium salt solution,
the basic phosphate salt solution, or both comprise ammonium
hydroxide.
5. The method of claim 1 wherein the non-polar immiscible solvent
is methylene chloride.
6. The method of claim 1 wherein the pH of the calcium salt
solution is about 10.
7. The method of claim 1 wherein the pH of the phosphate salt
solution is about 10.
8. The method of claim 1 wherein the at least one bioresorbable
polymeric material comprises a polylactic acid, a polyglycolic
acid, a poly(lactic acid-glycolic acid), a polyanhydride, a
poly(phosphazene), a poly(orthoester), a poly(caprolactone), a
polyhydroxybutyrate, a polyanhydrideco-imide, a polypropylene
fumarate, a polydiaxonane, or a polyurethane polymer, or any
copolymer or mixture thereof.
9. The bioresorbable composite of claim 1, wherein the
bioresorbable polymeric material comprises a polylactic acid, a
polyglycolic acid, a poly(lactic acid-glycolic acid) polymer or any
copolymer or mixture thereof.
10. The method of claim 1 wherein the temperature is below room
temperature.
11. The method of claim 1 wherein the temperature is about
-70.degree. C.
12. The method of claim 1 further comprising adding the emulsion to
a mixed solution of a surfactant and a calcium salt.
13. The method of claim 12 wherein the surfactant is poly(vinyl
alcohol).
14. The method of claim 1 further comprising separating the
encapsulated microspheres of bioresorbable polymer from
unencapsulated calcium phosphate and unencapsulating
microspheres.
15. The method of claim 1 wherein the calcium phosphate composition
is characterized by an x-ray diffraction pattern indicative of an
amorphous or poorly crystalline material.
16. The method of claim 1, wherein the calcium phosphate
composition is characterized as having a stoichiometry
approximately that of hydroxyapatite, calcium phosphate, tricalcium
phosphate, tetracalcium phosphate, bone apatite, or any combination
thereof.
17. The method of claim 1, wherein the calcium phosphate
composition is characterized as having a stoichiometry
approximately that of bone apatite.
18. The method of claim 1, wherein the calcium phosphate
composition additionally comprises carbonated calcium
phosphate.
19. The method of claim 1 wherein the bioresorbable microspheres
are characterized as having a diameter between about 100 and about
250 microns.
20. The method of claim 1 wherein the bioresorbable polymeric
microsphere contain approximately 28 weight percent or more of the
non-crystalline, poorly crystalline, or amorphous calcium phosphate
ceramic relative to the mass of the entire composite.
21. The method of claim 1 further comprising joining a plurality of
the encapsulating microspheres of the at least one bioresorbable
polymeric material to form a porous three-dimensional scaffold.
22. The method of claim 1 wherein the plurality of the
encapsulating microspheres of the at least one bioresorbable
polymeric material are joined by sintering at a temperature above
the melting point of at least one of the bioresorbable polymeric
materials.
23. The method of claim 22 wherein sintering temperature is about
150.degree. C.
24. The method of claim 21 wherein the majority of pores in the
three-dimensional scaffold are characterized as having diameters of
at least 100 microns.
25. The method of claim 15 wherein the porosity of the
three-dimensional scaffold is about 75%.
26. The method of claim 1 or 21 wherein the bioresorbable composite
is suitable for tissue repair and/or replacement applications.
27. The method of claim 26 wherein the tissue is bone.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation application of
application U.S. Ser. No. 10/469,617 filed Feb. 6, 2004, now
allowed, which is a National Stage Entry of PCT/US02/7854, having a
371c date of Mar. 14, 2002, which claims priority to application
U.S. Ser. No. 60/275,561, filed Mar. 14, 2001, each of which is
entitled "Polymeric Bioresorbable Composites Containing an
Amorphous Calcium Phosphate Polymer Ceramic for Bone Repair and
Replacement," and each of which is incorporated by reference.
TECHNICAL FIELD
[0003] The present invention relates to the use of biodegradable
composites for tissue engineering applications, particularly
methods for making the same.
BACKGROUND
[0004] With demographics shifting towards an older population and
with more people living more active lifestyles, the number of
orthopaedic injuries and disorders continues to rise. In the United
States alone, there were more than 6 million fractures each year
from 1992 to 1994 (Praemer, et al., American Academy of Orthopaedic
Surgeons 1999 182). In 1995, there were 216,000 total knee
replacements, 134,000 total hip replacements, and close to 100,000
bone grafting procedures performed. Traditionally, autografts and
allografts have been used by orthopaedic surgeons to repair
fractures and other bone defects. However, limitations including
donor-site morbidity, risk of disease transfer, potential
immunogenicity, and insufficient supply has led investigators to
search for alternative bone repair materials.
[0005] Since the main mineral component of bone is a complex
calcium phosphate system called apatite, hydroxyapatite and other
materials within the calcium phosphate family have been and
continue to be extensively investigated (DeMaeyer, et al. J.
Biomed. Mater. Res. 2000 52:95-106; Keller, L. and Dollase, W. A.
J., Biomed. Mater. Res. 2000 49:244-249; Zeng, et al. Biomaterials
1999 20:443-451; Ma, et al., J. Biomed. Mater. Res. 2001
54:284-293; and Duracan, C. and Brown, P. W. J., Biomed. Mater.
Res. 2000 51:726-734). Further, calcium phosphate ceramics have
been reported to be osteoconductive and to directly bond to bone
(Jarcho, M., Clin. Orthop. Rel. Res. 1981 157:259-278; Kitsugi, et
al., Clin. Orthop. Rel. Res. 1988 234:280-290). In addition,
calcium phosphate ceramics are believed to serve as precursors to
bone apatite formation in vivo. Accordingly, the good bone
compatibility of calcium phosphate ceramics is indicative of their
suitability for repair or replacement of damaged or diseased bone.
However, the brittleness of these materials limits their widespread
use in orthopaedics, particularly in load-bearing applications.
[0006] Accordingly, various attempts have been made to overcome
this limitation. One example has been to prepare composites of
these ceramics with bioresorbable polymeric materials such as
collagen and polymers of lactic acid and glycolic acid (TenHuisen,
et al., J. Biomed. Mater. Res. 1995 29:803-810; Yasunaga, et al.,
J. Biomed. Mater. Res. 1999 47:412-419; Zhang, et al., J. Biomed.
Mater. Res. 1999 45:285-293; Devin et al. J. Biomater. Sci. Polymer
Edn. 1996 7:661-669; Boeree et al. Biomaterials 1993 14:793-796).
In general, these composites are made porous in order to create a
3-dimensional scaffold that allows the ingrowth of new bone and the
eventual replacement of the scaffold with new skeletal tissue
(Zhang, et al., J. Biomed. Mater. Res. 1999 45:285-293; Devin et
al. J. Biomater. Sci. Polymer. End. 1996 7:661-669). In these
composites, the ceramic typically comprises a calcium phosphate
compound with moderate to high crystallinity (TenHuisen, et al., J.
Biomed. Mater. Res. 1995 29:803-810; Yasunaga, et al., J. Biomed.
Mater. Res. 1999 47:412-419; Zhang, et al., J. Biomed. Mater. Res.
1999 45:285-293; Devin, et al., J. Biomater. Sci. Polymer. Edn.
1999 7:661-669; Boeree et al. Biomaterials 1993 14:793-796).
[0007] In contrast, bone apatite is poorly crystalline and
non-stoichiometric due to the presence of other ions such as
magnesium and carbonate ions (Posner, A. S. and Betts, F., Acc.
Chem. Res. 1975 8:274-281; Bigi, et al., Calcif. Tissue Int. 1992
50:439-444). Further, crystalline forms of hydroxyapatite have been
shown to resorb at a slower rate than that of new bone formation.
In fact, the rate of new bone formation coincides more closely with
the resorption rate of poorly crystalline or amorphous calcium
phosphate ceramics (Frayssinet, et al., Biomaterials 1993
14:423-429; Klein, et al., J. Biomed. Mater. Res. 1983 17:769-784;
Knaack, et al., J. Biomed. Mater. Res. (Appl. Biomater.) 1998
43:399-409).
[0008] In the present invention, crystalline hydroxyapatite is
replaced with a poorly crystalline or amorphous calcium phosphate
ceramic believed to resorb concurrently with new bone growth. Also,
the degradation of the amorphous calcium phosphate forms alkali
products that serve to buffer the acidic degradation product of
either lactic or glycolic acid in a composite of the two
materials.
SUMMARY
[0009] An object of the present invention is to provide a
bioresorbable composite for use in bone repair and replacement
which comprises a non-crystalline or amorphous calcium phosphate
ceramic synthesized within encapsulating microspheres of a
bioresorbable polymeric material.
[0010] Another object of the present invention is to provide a
method for producing a bioresorbable composite which comprises a
bioresorbable polymeric material and a non-crystalline or amorphous
calcium phosphate ceramic for use in bone repair and replacement
wherein the method comprises synthesizing the calcium phosphate
ceramic within encapsulating microspheres of the bioresorbable
polymeric material.
[0011] Yet another object of the present invention is to provide
porous, 3-dimensional scaffolds with uniform composition throughout
the scaffold, wherein said scaffolds are produced by sintering
together microspheres of a non-crystalline or amorphous calcium
phosphate ceramic synthesized within encapsulating microspheres of
a bioresorbable polymeric material.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0012] In recent years, bioresorbable, porous, 3-dimensional
scaffolds have been studied as matrices for the regeneration of
tissues such as skin, cartilage and bone. The rationale for this
design is that the interconnected pores allow the ingrowth of new
tissue, which eventually replaces the degrading scaffold. In
orthopaedic applications, the scaffolds are typically made of
resorbable polymers, ceramics or composites of each.
[0013] The present invention relates to bioresorbable composites
which can be sintered into 3-dimensional scaffolds for bone repair
and replacement. These bioresorbable composites comprise a
non-crystalline or amorphous calcium phosphate ceramic synthesized
within an encapsulating microsphere of a bioresorbable polymeric
material.
[0014] Calcium phosphate ceramics useful in tissue scaffolds are
well known in the art. Examples of calcium phosphate ceramics which
can be used in the present invention include, but are not limited
to, hydroxyapatite, monocalcium phosphate, tricalcium phosphate,
and tetracalcium phosphate.
[0015] Various bioresorbable, biocompatible polymers have also been
created for use in medical applications. One of the most common
polymers used as a biomaterial has been the polyester copolymer
poly(lactic acid-glycolic acid) referred to herein as PLAGA. PLAGA
is highly biocompatible, degrades into harmless monomer units and
has a wide range of mechanical properties making this copolymer and
its homopolymer derivatives, PLA and PGA, useful in skeletal repair
and regeneration (Coombes, A. D. and Heckman, J. D., Biomaterials
1992 13:217-224; Mikos, et al., Polymer 1994 35:1068-1077;
Robinson, et al., Otolaryngol. Head and Neck Surg. 1995
112:707-713; Thomson, et al., J. Biomater. Sci. Polymer Edn. 1995
7:23-38; Devin, et al., J. Biomater. Sci. Polymer Edn. 1996
7:661-669). In a preferred embodiment of the present invention, the
bioresorbable polymeric material comprises PLAGA (50:50). However,
as will be understood by those of skill in the art upon this
disclosure, other polymeric materials including, but not limited
to, poly(anhydrides), poly(phosphazenes), poly(orthoesters),
poly(caprolactones), polyhydroxybutyrates, polyanhydride-co-imides,
polypropylene fumarates, polydiaxonanes, and poly(urethanes) can
also be used to form the encapsulating microspheres.
[0016] In the composites of the present invention, the calcium
phosphate ceramic is synthesized within the encapsulating
microspheres of the bioresorbable polymeric material. Thus, in the
present invention, the microspheres serve as micro-reactors for the
synthesis of the calcium phosphate ceramic. By carrying out the
synthesis within the confined space of the microsphere's interior,
crystal growth is impeded due to the constraints imposed by the
small internal space of the microsphere. As a result, the formation
of crystalline calcium phosphates is prevented or at least
minimized. Accordingly, the calcium phosphate ceramic of the
present invention more closely mimics the low crystallinity of bone
apatite and thus provides a better bone substitute than a highly
crystalline calcium phosphate material. In addition, amorphous or
low crystalline calcium phosphates have a faster resorption rate
than their crystalline counter parts, which allows them to be more
readily replaced by new bone. The degradation of an amorphous
calcium phosphate also forms alkali products that serve to buffer
the acidic degradation products of the bioresorbable polymeric
material of the microsphere. Inclusion of the bioresorbable
polymeric material, however, reduces the brittleness of the ceramic
component and serves to bind the microspheres together during
sintering of the microspheres of this composite into tissue
scaffolds. Additionally, because each individual microsphere is in
itself a composite, the resulting scaffold has uniform composition
throughout.
[0017] The reaction between calcium nitrate tetrahydrate and
ammonium hydrogen phosphate in basic aqueous solution has been
shown to produce an amorphous calcium phosphate, which becomes
crystalline over time (Eanes et al. Nature 1965 208:365-367). In
the present invention, a similar reaction was carried out within
microspheres made from the bioresorbable polymer, PLAGA.
Microspheres were prepared using a water-in-oil emulsion containing
PLAGA, calcium nitrate tetrahydrate, and ammonium hydrogen
phosphate. The water-in-oil emulsion was prepared by adding a basic
aqueous solution of the nitrate and phosphate components to a
solution of PLAGA in methylene chloride followed by rapid mixing.
The different components of the emulsion were cooled prior to
mixing in order to prevent the premature reaction of calcium
nitrate with the phosphate reagent which would result in
precipitation of a calcium phosphate compound at this stage of the
process. Once the emulsion was made, it was added to a solution of
poly (vinyl alcohol) containing an excess of calcium nitrate
tetrahydrate which was being stirred with a mechanical stirrer.
Excess calcium nitrate was added to minimize the diffusion of
calcium ions from inside the microspheres to the surrounding
solution, thereby increasing the chances of obtaining a calcium
phosphate with a high Ca/P molar ratio similar to that of bone
apatite, which has a Ca/P ratio of approximately 1.6 (Bigi et al.
Calcif. Tissue Int. 1992 50:439-444). The resulting microparticles
were collected after a 30-hour reaction period. These collected
microparticles consisted of the composite microspheres as well as
unencapsulated calcium phosphate particles. To separate the
composite microspheres from the unencapsulated ceramic particles,
the microparticle mixture was added to a hexane-water mixture,
which was then shaken and allowed to stand. This separation method
is based on the preference of the unencapsulated calcium phosphate
particles for the aqueous layer and the preference of the
microspheres for the organic layer due to the presence of the more
hydrophobic PLAGA in the microspheres. The hexane layer was then
collected and the microspheres isolated by filtration.
[0018] The ceramic content of the microspheres as determined by
gravimetric analysis, was approximately 28%. This can be increased,
however, if different mechanical properties are desired. For
example, ceramic content can be increased to increase compressive
modulus and other mechanical properties.
[0019] The synthesized calcium phosphate was characterized via
elemental analysis to determine its Ca/P molar ratio. Tetracalcium
phosphate was one of the products expected, the other being
hydroxyapatite. At pH 10, which was the pH of the reaction mixture,
both of these compounds are the least soluble of the various
calcium phosphates and are therefore more likely to form and
precipitate at this pH (Chow, et al., Mat. Res. Soc. Symp. Proc.
1991 179:3-24). Table 1 shows the % calcium (Ca), % phosphate (P),
and Ca/P ratio of the synthesized ceramic together with the
calculated values for commercially available hydroxyapatite and
tetracalcium phosphate.
TABLE-US-00001 TABLE 1 Ca and P analysis of synthesized calcium
phosphate and calculated values for known calcium phosphate
ceramics Elemental Analysis % Ca % P Ca/P molar ratio Synthesized
calcium 36.30 13.84 2.02 Tetracalcium 43.72 16.94 2.00
Hydroxyapatite 39.84 18.53 1.67
[0020] As shown, the Ca/P ratio closely matches that of
tetracalcium phosphate.
[0021] The synthesized material, as well as the commercially
available hydroxyapatite, was analyzed via infrared (IR). The IR
spectra for both compounds were very similar except that the
spectra of the synthesized calcium phosphate had peaks around
1400-1700 cm.sup.-1 indicating the presence of carbonate. These
peaks are similar to the ones observed for a carbonated apatite
that was obtained after implanting brushite for 2 weeks in the
femoral metaphysis of a rabbit (Constantz, et al., J. Biomed.
Mater. Res. (Appl. Biometer. 1998 43:451-461). Since bone apatite
has also been reported to contain substantial amounts of carbonate
(Posner, A. S. and Betts, F. Acc. Chem. Res. 1975: 8:273-281), the
synthesized calcium phosphate of the present invention more closely
resembles the composition of bone apatite than does tetracalcium
phosphate or hydroxyapatite.
[0022] To determine the crystallinity of the ceramic that was
synthesized, X-ray powder diffraction (XRD) was performed. From the
XRD spectra it could be seen that the hydroxyapatite was highly
crystalline while the synthesized calcium phosphate material
appeared non-crystalline. Further, the non-crystalline pattern was
similar to those seen in other amorphous calcium phosphate XRD
spectra (Eanes, et al., Nature 1965 208:365-367). This
non-crystalline pattern evidences the fact that by restricting the
synthesis of the calcium phosphate to the small space within the
microsphere's interior, crystalline formation was prevented. As
discussed, bone apatite is a poorly crystalline calcium phosphate
ceramic. Further, amorphous or poorly crystalline calcium
phosphates are more resorbable than their crystalline counterparts.
Thus, these results are indicative of the synthesized calcium
phosphate ceramic of the present invention being a suitable
material for bone repair when encapsulated within a bioresorbable
polymeric material such as PLAGA microsphere to offset its
brittleness.
[0023] The present invention also relates to 3-dimensional
scaffolds produced by sintering together microspheres of a
bioresorbable polymeric material and a non-crystalline calcium
phosphate ceramic synthesized within the microspheres. Various
conditions for sintering the microspheres can be used. Further,
such conditions can be routinely determined by those of skill in
the art upon reading this disclosure and based upon the melting
temperature of the bioresorbable polymeric material. For example,
composite microspheres comprising PLAGA were sintered at
150.degree. C. for 1 hour in order to fuse the microspheres
together. Fusion of the microspheres was possible because this
sintering temperature was above the melting temperature of PLAGA.
The resulting 3-dimensional scaffold was porous and did not crumble
upon handling unlike some sintered, porous, calcium phosphate
ceramics.
[0024] Image analysis of a cross-section of this scaffold by
scanning electron microscopy showed fused microspheres and deep
pores. In addition, some of the microspheres split open probably
due to the slight pressure provided by the piston of the
cylindrical mold assembly. As a result, the porosity of the
scaffold was increased further, which also increased the surface
area for cell attachment. Furthermore, the sintering process
transformed the surface of the microspheres into a rougher one,
characterized by the preponderance of micron-sized pores. Decades
ago, the minimum pore size for effective bone ingrowth into a
porous scaffold was established to be approximately 100 microns.
(Klawitter, J. J. and Hulbert, S. F., J. Biomed. Mater. Res. Symp.
1971 2:161; Gauthier, et al., Biomaterial 1998 19(1-3):133; and
Daculsi, G. and Passuit, N., Biomaterials 1990 11:86). Mercury
intrusion porosimetry on the scaffold indicated that a majority of
the pores have diameters of at least 100 microns, thus meeting the
minimum requirement for bone ingrowth. Porosimetry results also
showed that the scaffold had a porosity of approximately 75%.
[0025] Thus, as demonstrated herein, by restricting the reaction of
a calcium salt with a phosphate compound to the small confine of a
polymeric microsphere's interior, a non-crystalline, carbonated
calcium phosphate ceramic useful in combination with a
bioresorbable polymeric material as a composite for bone repair and
replacement was obtained. Further, sintering the composite
microspheres together produced a highly porous, 3-dimensional
scaffold with a rough surface. In all, the combination of
high-porosity and a calcium phosphate component that is
non-crystalline and carbonated renders the 3-dimensional composite
scaffolds produced from the composites particularly suitable for
bone repair and/or replacement applications.
[0026] The following non-limiting examples are provided to further
illustrate the present invention.
EXAMPLES
Example 1
Materials
[0027] Poly(lactide-co-glycolide) or PLAGA (50:50) with a molecular
weight of 50,000 was obtained from American Cyanamid. Calcium
nitrate tetrahydrate, ammonium hydrogen phosphate, and concentrated
ammonium hydroxide were obtained from Aldrich and used as received.
Commercial hydroxyapatite used for comparison was obtained from
Stryker Howmedica (Allendale, N.J.). Methylene chloride was
obtained from Fisher Scientific (Camden, N.J.) and used as
received.
Example 2
Preparation of Composite Microspheres
[0028] PLAGA (1.50 grams, 0.0115 mol) was dissolved in 9 mL of
methylene chloride. Calcium nitrate tetrahydrate (3.54 grams,
0.0150 moles) was dissolved in 1.5 mL of water previously adjusted
to pH 10 with ammonium hydroxide. In a separate vial, ammonium
hydrogen phosphate (0.99 grams, 0.0075 mol) was dissolved in 2.25
mL of water, pH 10. The polymer solution and the calcium nitrate
solution were kept at -70.degree. C. prior to use, while the
phosphate solution was maintained at room temperature. The cooled
nitrate solution was added quickly to the cooled polymer solution
followed immediately by the addition of the phosphate solution, and
vortex-mixed at high speed for 20 seconds to create an emulsion.
The emulsion was then poured in a slow steady stream into 600 mL of
a 1% polyvinyl alcohol (PVA) solution (pH 10) containing 30 grams
of calcium nitrate tetrahydrate which was being stirred at a speed
of 250 rpm during the addition and was raised to 500 rpm after the
addition. The reaction was allowed to proceed for 30 hours, during
which ammonium hydroxide was added at regular intervals in order to
maintain a pH of 10. Methylene chloride was allowed to evaporate
slowly during the reaction period. After 30 hours, the hollow
microspheres that floated to the top of the reaction mixture were
removed using a pipette. The microparticles that settled to the
bottom of the reaction vessel were collected by suction filtration
and washed several times with water, pH 10. The residue was
air-dried for 24 hours.
[0029] The air-dried residue was added to a reparatory funnel
containing 100 mL of a 50:50 mixture of water and hexane. The whole
mixture was shaken and allowed to stand for a few minutes. Most of
the encapsulated calcium phosphate particles settled into the lower
aqueous layer while most of the microsphere stayed in the upper
organic layer. The aqueous layer was removed and more water was
added and the separation process was repeated. This step was
repeated one more time and the microspheres in the organic layer
were collected by vacuum filtration, air-dried and then lyophilized
for 24 hours.
Example 3
Percent Ceramic Content
[0030] A known weight of microspheres was placed in methylene
chloride to dissolve away the PLAGA component. The mixture was
filtered and the residue placed in methylene chloride to remove
more PLAGA. The extraction step was repeated one more time. The
residue was collected, lyophilized and weighed. This experiment was
performed in triplicate.
Example 4
[0031] Characterization of Synthesized Calcium Phosphate
[0032] The calcium phosphate that was synthesized within the
microsphere was analyzed for calcium and phosphorus. Elemental
analysis was obtained from Quantitative Technologies, Inc.
(Whitehouse, N.J.). IR analysis was carried out using a Nicolet
Magna IR 560 (Madison, Wis.). Powder X-ray diffraction was
performed using a Siemens D500 diffractometer using Ni-filtered CuK
radiation with a 20 range from 2-60.degree. C.
Example 5
Preparation and Characterization of 3-Dimensional Scaffold
[0033] The composite microspheres (100-250 microns in diameter)
were placed in a 5-mm stainless steel cylinder mold and pressed
lightly with the corresponding sized piston. The whole assembly was
placed in an oven at 150.degree. C. for 1 hour in order to sinter
the microspheres. A cross-section of the resulting 3-dimensional
scaffold was viewed by scanning electron microscopy (SEM). Samples
were gold-coated using a Denton Desk 1 sputter coater in an
argon-purged chamber evacuated in 500 mTorr. The images were viewed
using an Amray 1830 D4 scanning electron microscope (20 kV) with a
tungsten gun and a diffusion pump. The porosity and the pore-size
distribution of the scaffold were determined using a Micromeritics
Autopore III Mercury Intrusion Porosimeter (Norcross, Ga.) by
infusing mercury into the samples with a pressure of 40 psi. Pore
size and porosity were determined as a function of pressure and
corresponding mercury intrusion volume. As the mercury is forced
into the pores of the sample, both the pressure necessary to move
the mercury and the volume of mercury moved are recorded. From
these data, both porosity and pore size are computed.
* * * * *