Note: Descriptions are shown in the official language in which they were submitted.
`11
FIELD OF T~E INVENTION
l The present invention is concerned with biocompatible
2 articles suitable ~or surgical intr~duction in-vivo and is par-
3 ticularly directed to bioerodible articles which degrade
4 predictably as biocompatible and non~toxic-products.
BACKGROUND OF THE INVENTION
S Synthetic polymeric compositions which are bioerodible
6 and biocompatible have become increasingly important and valuable
in recent years. One application for such compositions is as
8 surgically implantable biomaterials or prosthetic articlès ~or
9 human and animal subjects in-vivo. Consequently, such biomat-
erials axe articles which serve as implants, a tangible item
11 to be inserted into a living site as for growth or formation
12 of an organic union, or prostheses, artificial devices introduced
13 into living tissues to replace a missing part of the body;
14 these are exemplified by articles such as vascular grafts, bio-
degradable sutures and pellets, and orthopedic appliances such
16 as bone plates and the l~ke. In order for an implantable or
17 prosthetic article to be truly useful, it should be composed o~
18 a synthetic polymeric composition having specific characteristics
l9 and properties: First, the polymeric composition should have
a surface that permits and encourages growth of appropriate
21 mammalian cell types; that is, the growing and maintenance of
22 cells on, and i~ appropriate, within, its matrix, after intro-
23 duction into a subject _- iVO. For the purposes o~ graft mater-
24 ials, one objective is to maintain n o n - t h r o m b og e n i c
surfaces similar to that as exists in living tissues. A
~L~74~1L79
1 failure to initiate and maintain such en~othelial cell
2 surface layers leads to the occurrence of thrombotic
3 events such as occlusion of the blood vessel(s) an
4 ultimate failure of the implanted or prosthetic article.
Second~ the synthetic c~mposition should provide
6 sufficient elasticity and tensile strength over a
7 preselected minimal time period which will vary with the
8 specific application. Third, the synthetic composition
g should be non-immunogenic, biocompatible, biodegradable
in- ivo and yield degradation products which are
11 themselves non-inflammatory, non-toxic, and
12 non-antigenic. Lastly, the ideal material should
13 degrade within predictable periods of time and be
14 suitable for introduction at multiple tissue sites
thereby eliminating the need for surgical removal.
16 Despite continuing research efforts, no class of
17 synthetic polymeric biomaterials has yet been developed
18 which provides all these desired attributes. For
19 example, most of the research concerning synthetic
grafting materials has utilized compositions such as
21 Dacron [Graham et al., Arch. ~ . 115:929-933 ~1980);
22 Herring et al., Ann. sur~. 190:84-90 ~1979)]. These
23 materials, however, never develop the desired
24 endothelial cell intima necessary to maintain a
non-thrombo~enic surace; as a result, thrombosis -
26 blood vessel blockage and diminished blood cell supply
27 to organs in the body - often occurs. Similarly, the
28 use of polygalactin mesh has failed to provide the
29 necessary surface erosion characteristics and thus is
unpredictable in degradation time [Bowald et al.,
31 Surgerv 86:722-729 ~1979)]. Other presently known
I _ 3 _
1~ ` 12743L7~
1 . I biodegradable polymers such as polylactic acid,
i polyglycolic acid, polycaproloctones and the various
¦ polyamides also all degrade irregularly and
4 1 unpredictably with a demonstratable loss of permeability
and mechanical strength over time rHeller et al.,
6 ¦ "Theory and Practice of Control Drug Delivery rom
7 Bioerodible Polymers", in Controlled Release O
8 ¦ Bioactive Material, R.W. Baker Editors, Academic Press,
g I New York, 1980, pp. 1-17; Pitt et al., Biomaterials
¦! 2:215-220 (19811; Chu, C.C., J. ~ . PolYm. Sci.
26:1727-1734 (1981)]. Insoar as is presently known,
12 ,¦ therefore, no synthetic polymeric composition offers all
13 !¦ the properties and characteristi~s which would make it
14 ¦I desirable for use as an article for prosthesis or
il implantation in-vivo
I
SUMMARY OF THE_INVENTION
16 A bioerodible article useful for prosthesis and
17 implantation and methods for its manufacture are
19 .provided which comprises a biocompatible, hydrophobic
19 polyanhydride matrix, prepared in preselected dimensions
and configurations, which erodes predictably into
21 non-toxic residues a~ter introduction in-vivo. The
22 method o usiny the article as an implant and prosthesis
23 comprises the step of introducing a specifically
24 configured article into a subject in-vivo at a
¦ predetermined site.
. I DETAILED DESCRIPTION OF T~E DRAWING
26. The present invention may be more completely and
27 easily understood when taken in conjunction with the
28 accompanying drawing, in which:
.
-- 4 --
1~'74179
1 Fig. 1 is a graph illustrating the degradation
2 rates for poly [bis (p-carboxyphenoxy) propane
3 anhydride] matrices and its sebacic acid copolymer
matrices at 37C;
Fig. 2 is a graph illustrating the degradation
rates ~or compression molded poly tbis (p-carboxy-
7 phenoxy) propane anhydride] at different pH levels
8 xanging from 7.4 to 10.0;
g Fig. 3 is a set of two photographs illustrating the
biocompatibility attributes of the present invention
11 in-vivo as corneal implants in rabbits one week ater
12 introduction;
13 Fig. 4 is a photograph illustrating a magnified
14 view of the rabbit cornea of Fig. 3 in cross section;
Fig. 5 is a set of two photographs visualizing a
16 magnified view of rat skin tissue in cross section
17 ~ollowing a six month period of subcutaneous
18 implantation o~ the present invention: and
19 Fig. 6 is a set of two photographs visualizing a
magnified view o ~ixed poly~er-cell complexes cultured
21 in-vitro.
DETAILED DESCRIPTION OF THE PREFE~RED_E BODIMENTS
22 The present invention comprises articles useful as
23 implants or prostheses and methods for their preparation
24 and use. These articles comprise a biocompa~ible,
bioerodible, hydrophobic class of synthetic
26 polyanhydride polymeric compositions having the general
27 formula:
l ~
HC~ R--~H
wherein, R is a hydrophobic organic group and n is
2 greater than lo This class of polymeric compositions
3 can be formed in preselected dimensions and specific
4 . configurations. Regardless of the specific application,
. these compositions degrade withln predictable periods of
,~ time after introduction in-viYo into non-toxicr
non-inflammatory, and non-immunogenic residues.
The preEerred embodiments of the R group within the
general formulation given above is exemplified by, but
lo is not limited or restrict~d to, the entities given in
; 11 Table I below.
., . "'.
.
: TABLE I
whereln x > 2 and > 16
~b) , ~
(c)~ -C~
I . wherein 16 > x > 2
l (d) - ~ a ~ 2 ~
l wherëin x > 1
(e) ~~CH~ H2.CH;~ ~
wherein x > 1 and y > 1,
(f) ~ ~ ~ ~(0~
~herein R' and R- are organic groups
~ jl
I ~,7~
1 The entire class of polyanhydrides can be
2 synthesized using alternative methods of polymerization
3 now known in the art: bulk polymerization [Conix, A.,
4 Macro ~y~. 2:9598 (1966)3; solution polymerization
[Yoda et al., Bull. Chem. Soc. Japan 32:1120-1129
6 (1959)]; and interfacial polymerization [Matsuda et al.,
7 7apanese Patent No. 10,944 (1962)]. Using any of these
8 methods, a variety of different synthetic polymers
9 having a broad range of mechanical, chemical, and
10 ¦ erosion properties are obtained; the differences in
11 I properties and characteristics are controlled by varying
12 ¦ the parameters of reaction temperatures, reactant
13 concentration, types of solvent, and re~ction time.
14 This is true ~r all potential embodiments of R within
the general formula stated above as well as the entities
16 listed in Tl and the specific embodiments described in
17 the Examples which follow herein.
18 All of the articles useful as prostheses or
19 implants are synthetic polyanhydride polymeric
compositions which share a number of demonstratable
21 qualities in common:
2Z 1. Each of the articles displays ~redictable
23 degradation rates when introduced in-vivo in a subject.
24 ~egardless o the exact composition, size and
configuration, these articles erode only at their
26 exterior surfaces without affecting the center of the
~7 matrix in any way. As the surfaces continue to
28 pro~ressively erode, the articles become thinner and
29 smaller and eventually vanish complétely from the
tissue.
11 - 8 -
q4~g
1 Predictable times for degradation is a consequence
2 ' of biomaterial~ which erode by sur~ace (or
3 heterogeneous) erosion rather than by bulk (or
4 homog~neous) erosion. Surface erosion is degradation
which occurs only at the exterior surfaces of ~he
6 composition which, in turn, become progressively thinner
7 with the passage of time. Accordingly, by careful
8 selection oE the specific composition and control of the
g physical dimensions of the article, the user can
preselect predictable times for complete degradation to
11 occurO
12 2. The rate(s) of degradation for all entities
13 , within the class of polyanhydride polymeric compositions
14 as a whole is not only predictable, but may also be
controlled by varying the hydrophobicity of the polymer~
16 The mechanism of predictable degradation requires that ,
17 the polyanhydride polymers be hydrophobic in nature
18 thereby preventing water from entering into the interior
19 o~ the matrix in any appreciable degree. This may be
achieved alternatively either by substituting one
21 monomer (the R group) in the general formula for another
22 or by combining two monomerîc units as a copolymer and
23 then utilizing,the copolymer as the functional R group
24 within the composition. This is exempliied by the use
and attributes of the monomer poly (carboxyphenoxy)
26 propane alone and in combination with sebacic acid as a
2~ copolymer. Although each of these compositions contains
2~ individual hydrophobic properties~ each of these
29 polyanhydride polymerlc compositions contains
water-labile linkages between its monomer (or copolymer~
31 R units which ater introduction into the subject either
iL27~
l react with or become hydrolyzed by the subject's tissues
2 and body fluids.
3 3. The rates of degradation for each individual
4 poly~eric composition within the general class of
s polyanhydride polymers are predictable and constant at a
6 single pH level and present different and distinctive
7 rates of degradation with small changes of pH~ This
8 permits the articles to be ;ntroduced into the subject
9 at a variety o~ tissue sites; this is especially
lo valuable in that a wide variety of articles and devices
ll to meet diferent but specific applications may be
12 composed and configured to meet specific demands,
13 dimensions, and shapes - each of which offers
14 individual, but different, predictable periods for
degradation.
16 4. The entire class of polyanhydride polymers are
17 biocompatible and bioerodible. In view of their
18 intended function as an implant or prosthesis to be
l9 introduced into a subject in-vivo, it is absolutely
required that these compositions be non-inflammatory,
21 non-toxic, and non~immunogenic; that is biocompatible
22 with the subject's tissues and body fluids in all
~3 respec~s.
24 5. Implantable articles and prostheses formed of
polyanhydride polymers do not measurably affect or
26 influence living cells or tissues in any degree.
27 Various polyanhydride compositions may be combined with
28 large vessel endothelial cells and/or smooth muscle
29 cel7s without inhibiting or affecting cell growth.
In-vitro studies show that the endothelial or muscle
31 cells maintained a non-overlapping, contact-inhibited
l - 10 -
:"~`
1 monolayer of living cells throughout the testing period
2 of two weeks. Subsequently made histological studies of
3 these cultured specimens revealed attenuated endothelial
4 monolayers over the exterior sur~aces o~ the polymeric
matrices which strongly resemble intimal endothelial
6 monolayers in-vivo.
7 6. The articles comprising the present invention
8 degrade (erode~ into residues or moieties which are
9 themselves biocompatible and non-toxic. As evidenced by
~he Examples which ~ollow, each of the articles,
11 regardless of specific polyanhydride formulation used,
12 may be implanted into ~he cornea of rabbits without
13 causing inflammation even in a minor degree; this is in
14 stark contrast to presently known compositions (such as
polylactic acid matrices) which d em on s t r a te
16 at least minor inflammation of such corneal tissues.
17 Moreover, articles comprising polyanhydride compositions
18 are demonst~atably biocompatible as will be de~cribed in
19 a study involving subcutaneous implantation of such
articles in rats. Despîte their presence in the living
21 tissues over a period of weeks, no inflammatory cell
2~ infiltration (polymorphonuclear leukocytes, macrophages,
23 and lymphocytes) is s~en in the tissues adjacent to the
24 implant~ Equally important, as the article predictably
degrades, the degradation products are demonstrably
26 non~mutagenic, non-cytotoxic, and non-teratogenic.
27 It will be appreciated that these properties and
~8 ¦ characteristics, as well as the mechanical and chemical
29 attributes, identify and distinguish such articles as
being singularly suitable as implants or prostheses.
31 The Examples which ~ollow merely serve to illustrate one
32 or more o the above described characteris~ics, which
33 are representative o the entire class as a whole.
1.'~74~79
. ~
1 The articles prepared and tested were forms of poly
2 [bis (p-carboxyphenoxy) propane anhydride] ~hereinafter
"PCPP"~ and its copolymers with sebacic acid
4 (hexeinafter "PPCP - SA"~. These poly lbis (p carboxy-
S phenoxy) alkane anhydrldes] were synthesized by meit
6 polycondensation following the method of Conix [Macro
7 Synthe. 2:95-98 ~1966)]. Briefly summarized~ the
_ .
8 dicarboxylic acid monomers (or copolymers) were
9 converted to the mixed anhydride by total reflux in
acetic and anhydride. Caution was taken to avoid
11 excessive reaction, which would yield a highly insoluble
12 prepolymer difficult to purify; 30 minutes was deemed
13 sufficient. The prepolymers isolated were further
14 recrystallized in a 50:50 (v/v) mixed solvent of acetic
anhydride and dimethylformamide. A recrystallization
16 period of several weeks was sometimes necessary to
17 obtain a reasonable yield (30~)~ The prepolymers then
18 underwent melt polycondensation In vacuo under nitrogen
19 sweep. The prepolymers were prepared by reacting
different monomer ratios with acetic anhydride.
21 Attempts were also made to obtain the copolymers by
22 polycondensing the mixture o~ individually prepared
23 pxepolymers. In terms of controlling the final
24 composition and purity of the product, the latter
approach was found to be superior; however, because of the
26 difficulty of isolating the sebaci~ acid anhydride
27 prepolymer, the former method was deemed more conven-
28 ient. Regardless of the methodology used, the composi-
29 tion of the copolymers were determined by ultraviolet
spectrometry and weight analysis after decomposition in
31 1 M NaOH at 70C overnight.
-~2-
I
1 The resulting polymers were purified by extraction
with anhydrous ether in a Soxhlet E extractor for
3 several hours and then stored in a dessicator over
4 calcium chloride. The purified polymers, obtained as a
crystalline solid, were then ground in a Micro Mill
6 Grinder and sieved into a particle si~e ranging from
7 90-150 micrometers (hereinafter "um-). The polymer
8 particles were then pressed into circular disks using a
9 Carver Test Cylinder Outit at 30 ~PSI at 5C above the
polymer's glass transition temperatu;^e, TGV for ten
1~ ~ minutes. Those polymeric compositions that had glass
12 1 transition temperatures below 30C were molded at room
13 ¦ temperature. The dimensions of the circular disks were
14 14 millimeters ~hereinafter lmmn) in diameter,
O~9-lol mm thick, and weighed between 140 and 160
16 milligrams (hereinater "mg").
17 The degradation characteristics of such synthetic
18 polyanh-ydrides are demonstrated by hydrophobic disks
19 comprising PCPP; PCPP and PCPP-SA in 85:15 ratio;
PCPP-SA in a 85:15 ratio; PCPP-SA in a 45:S5 ratio; and
21 PCPP-SA in a 21:79 ratio. Circular disks comprising
22 each of these compositions were placed in 0.1 M
23 phosphate buffer, pH 7.4 at 37C for a time period of up
to 14 weeks. The results are given in Fig. 1 as
degradation profiles whose erosion kinetics were
26 followed by measuring the ultravio~et absorbance at 250
27 nanometers (hereinafter "nmn) of periodically changed
2a buffer solutions using a Perkin-Elmer W
29 Spectrophotometer model 553. As is readily seen, the
more hydrophobic polymers - PCPP and PCPP-S~ (85:15) -
31 displayed constant erosion kinetics over several months.
I.
Il - 13 -
~z~
l Moreover, as the sebacic acid content increased in the
2 polymeric composition, the polymers became more
3 hydrophilic and demonstrated increased rates of erosion.
4 The rate of erosion for the composition in fact
S increased 800 times when the sebacic acid content of the
polymer reached 80 percent~ It was also noted that the
7 more hydrophilic copolymer compositions, PCPP-5A (45:55)
a and (21:79~ tended to crumble in the later stages of
9 degradation.
lo A separate study investigated the affect of
11 increasing the size of the alkane monomer and its affect
12 on degradation rate in the homologous poly [bis
13 (p-carboxyphenoxy) alkane] seriesO It was found that as
14 the number of methylene units in the R group backbone
was increasedr the polymeric composition became more
16 hydrophobic and the degradation rates decreased several
1~ orders of magnitude. Specifically, as the methylene
18 units were increased from 1 to 6 in the composition, the
19 degradation rate underwent a decrease of 3 orders of
magnitude. By extrapolation of the data in ~ig. 1, the
21 PCPP polymer is predicted to degrade completely and
22 slightly over 3 years' time; it is also thereore
23 apparent that the predictable time for degradation can
24 be increased or decreased at ~lill by several orders of
magnitude by either increasing or decreasing the alkane
26 size.
Example_2
27 The degradation rates for PCPP at a variety of pH
28 levels ranging from 7.4-lO.0 were determined~ The PCPP
29 was prepared, purified, and compression molded into
~ - 14 -
1 circular disks as described earlier in Example 1. Each
2 disk was placed in 10-50 milliliters (hereinaf~er ~mln)
3 of phosphate buffer held at 37~C which was prepared at
4 specific pH levels. The erosion of the disk was again
followed by W absorbance at 250 nm over a test period
6 of 350 hours. The results are illustrated in Fig~ 2
7 which demonstrates that the rate of degradation is
8 increased by a ~actor o~ 20 as the pH is increased fr~m
9 7.4 to 10Ø It is also noteworthy that the rates of
degradation remain stable and constant during the entire
11 two wee~ testing period.
Example 3
12 The biocompatibility and toxological effects were
13 investigated using the representative polyanhydride
14 polymers PCPP, PCPP-SA (45:55) poly (terephthalic acid
S anhydride) hereinafter "PTA"~ and poly
16 (terephthalic-sebacic acid anhydride) hereinafter
17 "PTA-SA" in a 50:S0 ratio. The polymers PCPP and
18 PCPP SA ~45:55) were prepared in the manner described in
19 Example 1. PTA and PTA-SA (50:50) were synthesized in
solution by an adaptation o~ the method of Yoda and
21 Matsuda tBull. Chem. Soc~ Japan 32:1120-1129 (1959);
22 J~panese Patent No. 10944]. This technique of solution
23 polymerization utilizes a dehydrative coupling reaction
24 between an acyl chloride and a carboxyl group to obtain
the polymerl This polymerization technique is
26 pre~errable because the high melting point (372C) of
27 the PTA polymer and its instability (charring) at ~his
28 temperature made the melt-polycondensation methodology
29 unsuitable. Preferrably, 0.02 mole of terephthalic acid
l - 15 -
~ ~4~.7~
,
1 (or 0.02 mole of sebacic acid) was dissolved in 400 ml
2 of chloro~orm in the presence of 0.04 mole of
triethyl~mine. Terephthaloyl chloride (0.0~ mole3
4 previously dissolved in benzene was added through a
dropping funnel over a 30 minute period under vigorous
6 agitation. This mixure was allowed to react for three
7 ! hours at room temperature and was held under a nitrogen
8 ¦ sweep at all times. The resulting polymer, PTA or
9 PTA-SA (50:50) was then purified by extraction with
I anhydrous ether in a Soxhlet Extractor for ~-3 hours and
11 was then stored in a dessicator over calcium chloride.
12 1 All samples for toxicological studies were prepared
13 ~ by placing each polymer ~n O.l M phosphate buffer,
14 1 pH 7.4, ~or several days and allowing the polymer to
1 degrade. The concentration of the degradation products
16 , was then determined by ultraviolet spectrophotometry.
17 ¦ The cytotoxicity and mutagenicity of the respective
1~ degradation products for the copol~mer PCPP-SA (45:55)
19 were determined by a forward mutation assay in
Salmonella typhimurium using 8-azaguanine resistance as
21 a genetic marker as described in Skopec et al., Proc.
22 NatlO Aca. Sci. U~S.A. 75:410-414 (1978). This
23 mutagenicity assay also includes a test for toxicity so
2~ that the mutagenicity, i~ present, can be expressed
quantitatively as the number of mutants per surviving
26 cell. In this way, an independent measurement of
27 toxicity for this bacterial species can be obtained.
28 Samples of PTA-SA (50:50) were tested at 1 mg/ml both
29 with and without the addition of mammalian metabolism
enzymes. The results o this assay demonstrated that
31 j the degradation products of this polymer were
I
Il - 16 -
1274179
1 non-mutagenic with or without the addition of a
2 mammalian metabolizing system. In ail instances, the
3 induced mutant fraction was essentially 0 being
indistinguishable from the spontaneous background
! control. Similarly, there was no toxicity in any
6 ¦ sample ~ithout the addition of metabolic enzymes and a
7 slight, but not significant, toxicity in samples with
8 the metabolizing system~ ¦
g The teratogenic potential was measured using a
¦ newly developed in-vitro assay in which the attached
11 efficiency of ascetic mouse ovarian tumor cells to
12 plastic surfaces coated with concanavalin A was
13 1 determined [Braun et al., Proc. Natl. Aca. Sci~ U.S.A.
14 79:2056-2060 (1982)]. In general with this assay,
non-teratogens do not inhibit attachment of the tumor
16 cells to the plastic surfaces. The degradation products
17 of each respective polymer in phosphate buffer solutions
18 was titrated to p~l 7.4 with NaOH before ~the tests were
19 conducted since the attachment is sensitive to acidic pH
levels. The reaction mixture of degradation products
21 and tumor cells was allowed to react for 2 hours at room
22 temperature and a cell suspension was bufered with 50
23 mM of HEPES bufer during the two hour incubation
24 period. The preferred concentration for each sample was
2S 0.035 mg/ml. The teratogenicity test results indicated
26 an average decline in attachment efficiency of 35 -~3~.
27 Given that the criteria for potential teratogenicity is
28 an inhibition of cell attachment by more than 50%, the
29 degradation products of these polyanhydr;de compositions
are considered non-teratogenic.
,
- 17 -
` 11 lZ~41~9
1 Testing in-vivo for the localized tissue response
2 to the polymers was determined by implantation in the
3 corneas of rabbits and subcutaneous introduction in
4 rats. For this series of tests, PCPP and PTA 5A (50:S0)
S were fashioned into pellets whose dimensions were 1 mm x
6 1 mm x 0.5 mm and implanted surgically in rab~it
7 intracorneal pouch~s within the corneal stroma following
8 the methodology described in Langer et al., J. Biomed.
g Mat. Res. 15:267-277 (1981). The rabbit corneas were
then inspected twice weekly by stereomicroscopy for
11 signs of inflammation as manifested by edema, cellular
12 infiltration, and neovascularization for a period of six
13 weeks. The host response to the polymeric pellets
14 implanted in the rabbit corneas is seen in Fig. 3a and
3b. Fig. 3a is the stereomicroscopic observation of the
16 cornea one week after implantation; Fig. 3b shows the
17 same cornea after the polymeric pellet had complet~ly
18 degraded after six weeks. No inflammatory response or
19 characteristics were observed at any time over the
entire six week implantation and degradation period;
21 urthermore, the clarity o~ the corneas was maintained
22 and the prolife~ation oE new blood vessels absent in all
23 instances. In addition, a~ter the six week testing
24 period, each cornea was surgically removed and prepared
~or histological examination to confirm and verify the
26 accuracy of the stereomicroscopic data. A
Z7 representative cross section of rabbit cornea is seen in
28 Fig. 4 which provides a highly magnified view of the
29 epithelial cell layer EP, the cornneal stroma ST, and
the endothelial cell layer EN. As is apparent there is
31 a total absence of inflammatory cells throughout the
32 corneas and this tissue appears normal in all respects.
` 1;2~ 7~
1 The in-vivo tissue response was determined by
2 subcutaneously implanting RCPP pellets in the abdominal
3 region of Sprague-Dawley rats following the procedure o
4 Brown et al., J. Pharm. Sci~ 72:1181-1185 (1983)~ After
surgical introduction of the polymer pellet into the
6 rat, the animals were maintained normally for a six
7 month period ~ithout special attention. The rats were
8 then sacrificed and histological sections of the
g abdominal tissues surrounding the site of implantation
prepared and studied. The results are illustrated in
11 Fig. 5a and 5bo Fig. 5a is a magnified cross-sectional
12 view of the abdominal muscle wall in cross section
13 showing s~eletal muscle and blood vessels; Fig. 5b is
14 the identical tissue seen at a greater degree of
magnification. It is apparent in each instance that no
16 inflammatory cell infiltration, that is the appearance
17 of polymorphonuclear leukocytes, macrophages and
18 lymphocytes, is seen in the tissues adjacent to the
19 implantation site. Similarly, gross post mortem
inspection also did not reveal any abnormalities of any
21 kind at the împlantation site.
Example 4
22 To demonstrate the biocompatibility and non-toxic
23 properties of polyanhydride polymeric compositions as
24 a whole, additional in-vitro tissue culture studies were
performed using PCPP-SA (45:55), PTA-SA (50:50) and PTA,
26 each of which was prepared as previsouly described.
21 The tissue culture studies relied on the ability of
28 endothelial cells and smooth muscle cells to grow and be
29 maintain in culture on t e s~rfsce of these polymers.
'9_ '
~1274179
1 Endothelial cells were isolated from bovine aortas by
2 collagenase digestion and then cultured in-vitro using
3 the procedures described in Jaffe et al., J. Clin~
4 Invest. 52:2745-2756 (1973~. The identity of these
endothelial cells was confirmed by staining ~or ~he
6 presence of Factor VIII antigen lJaffe et al., J. Clin.
7 Invest. 52:2757-2764 (1973). Smooth muscle cells were
8 obtained ~y explantation oE bovine aortic medial tissue
9 ¦ following the methods of Ross, R., J. Cell. Biol.
lo 50:172-186 (1971). At confluence, each cell type grew
11 in a char~cteristic "hill and valleyr morphology.
12 Approximately 1.0 x 104/cm2 cells which had been
13 previously passed in culture 4-15 times were plated
14 directly onto the circular pieces of pol~mer (having
dimensions of approximately 1.5 cm2 x 1 mm) in a 0.25 ml
16 drop of culture medium and allowed to react for one
17 hour's duration at room temperature~ The polymeric
18 disks were then flooded with 15 ml of Dulbeccio's
19 Modified Eagle's medium containing 10% calf serum in
100 mm containers. The culture medium was changed daily
21 to avoid accumulation of the acidic polymer degradation
22 products. After two weeks in culture at room
23 temperature, the polymeric disks and adheren~ cells ~ere
24 rinsed with phosphate buffered saline, pH 7.2, and fixed
~or one hour with a 1~ buffered glutaraldehyde solution.
26 Examination of the cultured cells and polymers
27 reveal the following: the bovine aortic endothelial
28 cells grew normally both over the surfaces of the
29 polymers and in the Petri dish containing media and the
degradation products of the polymer. In both instances
31 the cells displayed a normal morphology of polygonal
I - 2~ -
1 cells in a monolayer conformation typical of endothelial
2 cells. ~here was no evidence of any toxic ef~ect 011
3 these cells whatsoever~ Similarly, smooth muscle cells
4 also grew normally over the surfaces of the pol~mers and
in the presence of the degradation products. These
6 smooth muscle cells were examined also for abnormalities
7 on the basis of typcial cell morphology or inhibition of
8 their ability to proliferate. Deleterius effects we're
g absent and growth was normal for all samples tested.
Moreover, it was found that both the endothelial
11 cells and the smooth muscle cells grew and reproduced in
12 approximately the same amount of time in the control
13 samples utilizing polystyrene tissue culture plastic and
14 using the polyanhydride polymeric substanceO The
1S doubling times for endothelial cells were 16 ~2.5 hours
16 and 15 ~3.0 hours for the polystyrene plastic and the
11 polyanhydride polymers respectively: similarly, the '~
18 doubling,growth times for smooth muscle cells was 8 +1.5
19 hours and 8 ~2.0 hours using smooth muscle cells in the
polystyrene plastic and polyanhydride polymers
21 respectively. Gross visual observation of the
22 attachment of both kinds o~ cells to the polyanhydride
23 polymers was dificult if not impossible because of the
24 opa~ue character of the polymers. For this reason,
histiological sections were prepared and stained to
26 , confirm the normal appearance and growth of each type of
27 cell. Cross-sections o~ the fixed polyanhydride
28 polymer-complexes are seen in Figs. 6a and 6b which
29 reveals the presence of growing endothelial cells a~ a
flattened monolayer similar in character and appearance
31 to endothelial cells growth as an intima layer on blood
I - 21 -
~Z741~9
1 vessels in-vivo. There is no evidence of any enlarged
2 cells, there are no vacuoles, and there is no loss of
3 the normal growth pattern which would be visible if cell
growth were co~pact-inhibited by the polyanhydride test
s samples or their degradation products~
6 Bioerodible and biocompatible polyanhydride
7 polymeric compositions may be prepared ;n a wide variety
8 of specific formulations and be formed into preselected
9 dimensions and desired configurations as articles useful
lo for implantation or prosthesis. Any of the presently
11 known compression molding techniques? casting
12 techniques, or other manufacturiny processe~ may be used
13 to fashion the article into the desired dimensions and
14 configurations to meet the specific application. The
variety and multiplicity of uses for such articles are
16 exemplified by vascular graft materials, bioerodible
17 sutures, artificial skin surfaces, and orthopedic
18 devices generally such as bone plates and the like. In
19 particular applications the articles may also contain
additional substances such as collagen, elastin,
21 proteins and polypeptides which also aid and promote
22 healing and repair o body tissues and organs.
23 A~ter the articles have been ~ormed into the
24 desired dimensions and configurations, it is introduced
into the subject _-vivo at a predetermined tissue site~
26 It is expected that presently known surgical procedures
27 will be used for introduction of the article as these
23 are presently the most efficient and most reliable
29 techniques. In certain instances it can be seen that
alternate methods of introduction will also be useful~
31 The speci c site for introduction of the article is
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` ~ ~7
a matter of the user's need or choice and may be either
2 subcutaneous, superficial, or deeply imbedded into the
3 tissues or organs of the sub~ect as the need arises. It
~ will be recognized that the precise method of
introduction and the particular site of implantation are
6 of no consequence and are merely selected at will from
7 the many procedures and applications suitable for use.
8 The present invention is not to be restricted in
9 form nor limited in scope except by the claims appended
hereto.
'. .'
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