U.S. patent application number 15/604795 was filed with the patent office on 2017-09-14 for resorbable cellulose based biomaterial and implant.
The applicant listed for this patent is DePuy Synthes Products, Inc.. Invention is credited to Wojciech Czaja, Dmytro D. Kyryliouk.
Application Number | 20170260297 15/604795 |
Document ID | / |
Family ID | 47833418 |
Filed Date | 2017-09-14 |
United States Patent
Application |
20170260297 |
Kind Code |
A1 |
Czaja; Wojciech ; et
al. |
September 14, 2017 |
RESORBABLE CELLULOSE BASED BIOMATERIAL AND IMPLANT
Abstract
The present disclosure describes an implant for tissue
replacement or augmentation including a resorbable non-pyrogenic
porous body of irradiated oxidized cellulose, formed from a
precursor reactive mixture of irradiated cellulose and an oxidizing
agent, where the body forms a heterogeneous three-dimensional
fibrillar network.
Inventors: |
Czaja; Wojciech;
(Downingtown, PA) ; Kyryliouk; Dmytro D.;
(Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DePuy Synthes Products, Inc. |
Raynham |
MA |
US |
|
|
Family ID: |
47833418 |
Appl. No.: |
15/604795 |
Filed: |
May 25, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14735591 |
Jun 10, 2015 |
9670289 |
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15604795 |
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13773923 |
Feb 22, 2013 |
9090713 |
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14735591 |
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61601653 |
Feb 22, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08B 15/02 20130101;
A61L 27/20 20130101; A61L 31/148 20130101; A61L 31/042 20130101;
A61L 27/58 20130101; A61L 27/20 20130101; C08L 1/04 20130101; A61L
31/042 20130101; C08L 1/04 20130101 |
International
Class: |
C08B 15/02 20060101
C08B015/02; A61L 27/58 20060101 A61L027/58; A61L 27/20 20060101
A61L027/20 |
Claims
1. (canceled)
2. An implant for soft tissue repair comprising: an oxidized film
of irradiated microbial cellulose; wherein the film is porous and
resorbable; wherein the film has a hydrated state upon exposure to
fluid; and, wherein the film in the hydrated state is configured to
conform to a soft tissue surface.
3. The implant of claim 2, wherein the film has a cellulose content
in the range of about 2 mg/cm.sup.2 to about 15 mg/cm.sup.2.
4. The implant of claim 2, further comprising an active agent.
5. The implant of claim 4, wherein the film is configured as a
scaffold for the active agent.
6. The implant of claim 4, wherein the active agent is impregnated
within the film.
7. The implant of claim 4, wherein the active agent is coated onto
the film.
8. The implant of claim 2, wherein the film comprises an active
agent impregnated within the film, and wherein the film comprises
an active agent coated onto the film.
9. The implant of claim 2, wherein the film in the hydrated state
is configured to self-adhere to the soft tissue surface.
10. The implant of claim 2, wherein the implant is configured as a
hemostat.
11. The implant of claim 2, further comprising a secondary
implantable device.
12. The implant of claim 11, wherein the film is configured as a
hemostat.
13. The implant of claim 2, wherein the film has an in vitro
degradation rate in one week under simulated body fluid (SBF)
conditions of about zero percent to about 90 percent.
14. The implant of claim 2, wherein the film has an in vitro
degradation rate in four weeks under simulated body fluid (SBF)
conditions of about 80 percent to about 100 percent.
15. The implant of claim 2, wherein the film has an in vitro
degradation rate under simulated body fluid (SBF) conditions such
that the film remains mechanically stable at least between 2 weeks
to four weeks.
16. The implant of claim 2, wherein the film, in the hydrated
state, has a burst strength in the range of about 3 N to about 30
N.
17. A method for repair of a soft tissue comprising: attaching a
porous oxidized film of irradiated microbial cellulose to a surface
of a soft tissue, wherein the film has a hydrated state upon
exposure to fluid; wherein attaching the film in the hydrated state
to the surface of the soft tissue conforms the film to the surface
of the soft tissue; and, wherein, after attaching, the film in the
hydrated state is self-adhering to the surface of the soft
tissue.
18. The method of claim 17, wherein the film is configured as a
hemostat.
19. The method of claim 17, wherein the film further comprises an
active agent.
20. The method of claim 19, wherein the active agent is impregnated
within the film.
21. The method of claim 19, wherein the active agent is coated onto
the film.
22. The method of claim 17, wherein the film is resorbable, and has
an in vitro degradation rate in four weeks under simulated body
fluid (SBF) conditions of about 80 percent to about 100
percent.
23. The implant of claim 17, wherein the film, in the hydrated
state, has a burst strength in the range of about 3 N to about 30
N.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of co-pending U.S.
application Ser. No. 14/735,591, filed Jun. 10, 2015, which is a
continuation of U.S. application Ser. No. 13/773,923, filed on Feb.
22, 2013, now U.S. Pat. No. 9,090,713, which claims priority to
U.S. Provisional Application Ser. No. 61/601,653, filed Feb. 22,
2012, the disclosures of which are hereby incorporated by reference
as if set forth in their entirety herein.
FIELD OF THE DISCLOSURE
[0002] The present disclosure relates to a resorbable, porous and
conformable biomaterial for use as a medical implant and a
controlled oxidation process of .gamma.-irradiated cellulose to
provide the same. The implant can be formed as a sheet or patch for
use in tissue replacement or augmentation, particularly for soft
tissue indications and more particularly for use with dura
mater.
BACKGROUND
[0003] Repair of the dura (duraplasty) is indicated following
traumatic, neoplastic, or inflammatory destruction, surgical
excision, or congenital absence. Dural replacements are used in
cranial surgery when primary closure of native dura is not
possible. Historically, numerous materials have been used including
metal foils, human tissues, animal tissues (porcine dermis, bovine
collagen and pericardium) and polymers (PTFE, polyglactin,
hydroxyethylmethacrylate). Animal tissues remain the best of the
currently available materials with bovine pericardium and bovine
collagen being the market leaders (e.g., Duragen.RTM.,
Duraform.RTM.). However, the animal material carries the
possibility of infection by prions that may cause mad cow disease.
Also, bovine collagen often resorbs within two weeks, prior to
complete healing of the dura. Additionally, bovine pericardium is
sometimes cross-linked with glutaraldehyde, which has natural
biotoxicity. Synthetic materials have handling deficiencies and may
cause cerebrospinal fluid (CSF) leakage if not properly sutured in
place.
[0004] Cellulose of various origins has been proven to be a
versatile biomaterial. Synthesized by just about every type of
plant and a select number of bacteria, it is a natural, renewable,
biocompatible, and biodegradable polymer used in a wide variety of
applications.
[0005] However, native cellulose cannot be resorbed in human body
due to the lack of enzymatic machinery able to break down its
highly crystalline structure, which is stabilized by inter and
intra hydrogen bonds. Resorbability of cellulose can, however, be
achieved through oxidation using various chemicals, including
metaperiodate, hypochlorite, dichromate, or nitrogen dioxide (see
Stilwell et al., Oxidized cellulose: Chemistry, Processing and
Medical Applications, Handbook of Biodegradable Polymers: 1997,
291-306.). Oxidized plant cellulose has been successfully used as a
resorbable hemostat (Johnson and Johnson's Surgicel.RTM. since 1949
and more recently by Gelita Medical's Gelitacel.RTM. since 2006).
Products consisting of plant based oxidized cellulose are commonly
used as hemostatic agents, wound dressings and anti-adhesion
barriers (see U.S. Pat. No. 6,800,753; Stilwell et al., 1997).
[0006] Plant cellulose is oxidized most effectively through the use
of nitrogen dioxide gas vapor. However, there are toxic effects to
be considered from the use of nitrogen dioxide gas; whereas sodium
metaperiodate has proven to be more selective when oxidizing highly
crystalline celluloses with minimal side reactivity (see Nevell T.,
Oxidation, Methods in Carbohydrate Chemistry, New York: Academic
Press 1963; 3: 164-185). Its oxidizing effects and methods of use
have been studied extensively on plant cellulose (see Stilwell et
al., 1997; Kim et al., Periodate oxidation of crystalline
cellulose, Biomacromolecules 2000; 1: 488-492; Calvini et al., FTIR
and WAXS analysis of periodate oxycellulose: Evidence for a cluster
mechanism of oxidation, Vibrational Spectroscopy 2006; 40:
177-183.; Singh et al., Biodegradation studies on periodate
oxidized cellulose, Biomaterials 1982; 16-20; Devi et al.,
Biosoluble surgical material from 2,3-dialdehyde cellulose,
Biomaterials 1986; 7: 193-196.; Laurence et al., Development of
resorbable macroporous cellulosic material used as hemostatic in an
osseous environment, J Biomed Mater Res 2005; 73A: 422-429;
Roychowdhury and Kumar, Fabrication and evaluation of porous
2,3-dialdehyde cellulose membrane as a potential biodegradable
tissue-engineering scaffold, J Biomed Mater Res 2006; 76A:
300-309.). The mechanism of oxidation using periodate relies on
cleavage of the C2-C3 bond in the glucopyranose ring and formation
of dialdehyde groups. Such a dialdehyde cellulose is believed to
degrade by hydrolysis under physiological conditions seen in the
body into 2,4-dihydroxybutyric acid and glycolic acid (see Singh et
al, 1982). Both of these degradation products are known to be
biocompatible and biodegradable and can be metabolized by the body
(see Devi et al., 1986; Singh et al., 1982). Once the degradation
process is initiated it continues along the glucan chains that
comprise the cellulose network (see Stilwell et al., 1997).
[0007] Methods for oxidation of bacterially-derived cellulose have
also been described in U.S. Pat. No. 7,709,631. Bacterially-derived
cellulose possesses unique physical and mechanical properties which
results from its three-dimensional structure. Due to its handling
characteristics, biocompatibility, and safety, it is already used
in several medical devices, for example as described in U.S. Pat.
Nos. 7,374,775 and 7,510,725. One type of microbial cellulose
synthesized by Acetobacter xylinum (reclassified as
Gluconacetobacter xylinus) is characterized by a highly crystalline
three-dimensional network consisting of pure cellulose nanofibers.
Microbial cellulose has long been recognized as a biomaterial with
potential applications for temporary wound coverage, for treatment
of chronic wounds and burns, and as a scaffold for tissue growth,
synthetic blood vessels, as well as many other biomedical
applications (Fontana et al., Acetobacter cellulose pellicle as a
temporary skin substitute, Appl Biochem Biotechnol 1990; 24/25:
253-264; Alvarez et al, Effectiveness of a Biocellulose Wound
Dressing for the Treatment of Chronic Venous Leg Ulcers: Results of
a Single Center Random, Wounds 2004; 16: 224-233; Czaja et al., The
future prospects of microbial cellulose in biomedical applications,
Biomacromolecules 2007; 8(1): 1-12; Klemm et al., Cellulose:
Fascinating Biopolymer and Sustainable Raw Material, Angew Chem,
Int Ed 2005; 44: 3358-3393; Bodin et al., Bacterial cellulose as a
potential meniscus implant, J Tissue Eng and Regen Med 2007; 1(5):
406-408; Svensson et al., Bacterial cellulose as a potential
scaffold for tissue engineering of cartilage, Biomaterials 2005; 26
(4): 419-431).
[0008] Although methods for oxidizing cellulose are widely
described in the literature they often do not result in
homogenously oxidized materials with the most desirable properties
for medical applications. It is particularly true for soft tissue
applications, for example dural repair applications, where the
material needs to be able to rehydrate, readily conform to the
various contours of the body, have adequate strength to allow easy
handling, but also to be resorbable over a time frame that is
compatible with healing of the particular anatomical site.
Consequently there is a need for oxidized cellulose biomaterials
and methods for producing the same that can achieve these desired
properties.
[0009] The ideal material should be able to prevent CSF leakage,
have good biocompatibility, be free of potential risk of infection,
have good intra-operative handling, have mechanical properties
similar to dura, have a resorption profile beneficial to tissue
regrowth, and be readily available and storable.
SUMMARY
[0010] The present disclosure describes an irradiated oxidized
cellulose for use as a resorbable biomaterial that is formed from a
precursor reactive mixture of an irradiated cellulose and an
oxidizing agent. The reaction product thereof is a resorbable
biomaterial that is non-pyrogenic and can be porous. According to
one embodiment, the irradiated cellulose is microbial-derived
cellulose, and in a preferred embodiment is derived from
Gluconacetobacter xylinus. The resorbable biomaterial as described
can have a variable range of degree of oxidation, which can,
according to one embodiment, be in the range of about 0 percent to
about 99 percent oxidation, for example in the range of about 20
percent to about 70 percent.
[0011] The present disclosure additionally describes a medical
implant for use in tissue repair, replacement or augmentation
formed from a porous body of irradiated-oxidized cellulose, that,
according to one embodiment, can be formed by reacting irradiated
cellulose with an oxidizing agent. The oxidized cellulose body that
forms the implant has a chaotic, heterogeneous three-dimensional
fibrillar network that can allow the implant to rapidly transition
from a first rigid (dehydrated) state to a second hydrated state
upon contact with biocompatible fluids (e.g., water, saline, blood,
cerebrospinal fluid etc.). The implant in the hydrated state can,
according to one embodiment, have a surface that is conformable to
an anatomical surface, preferably a soft tissue surface, and more
preferably to a dural tissue surface. According to another
embodiment, the surface of the implant can be conformable to a
secondary medical device. According to a further embodiment, the
implant can be a scaffold or carrier for an active agent. For
example, the active agent can be impregnated within the porous body
of the implant, or coated onto a surface of the implant, or both.
According to one embodiment, the active agent can be impregnated
within and/or coated onto the implant substantially at or near the
time of implantation (i.e., intraoperatively). In an alternative
embodiment, the active agent can be impregnated within and/or
coated onto the implant prior to the time of implantation (i.e.,
preoperatively). In certain embodiments, more than one active agent
can be impregnated within and/or coated onto the implant, and
further the more than one active agents can be impregnated within
and/or coated onto the implant at different time periods. For
example, some active agents can be preoperatively combined with the
implant, while other active agents can be combined
intraoperatively.
[0012] The present disclosure further describes a method of
producing a body of oxidized cellulose that is porous and
resorbable including:
[0013] (a) irradiating a body of cellulose so as to form an
irradiated body of cellulose, and
[0014] (b) reacting the irradiated body of cellulose with an
oxidizing agent so as to form a body of oxidized cellulose.
[0015] The body of oxidized cellulose formed can be, according to
one embodiment, porous, non-pyrogenic, and resorbable.
[0016] According to one embodiment, the method can further include
the step of partially dehydrating the body of irradiated cellulose,
preferably by mechanically pressing the cellulose body. According
to another embodiment, the method can further include the step of
at least partially dehydrating the body of oxidized cellulose,
preferably by critical point drying using supercritical carbon
dioxide. According to an additional embodiment, the step of
irradiating the non-pyrogenic body can include one, or
alternatively more than one, doses or exposures of radiations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The drawings illustrate generally, by way of example, but
not by way of limitation, various embodiments discussed in the
present document. The foregoing summary, as well as the following
detailed description of preferred embodiments of the application,
will be better understood when read in conjunction with the
appended drawings.
[0018] FIG. 1 is a graphical depiction of proposed in vivo
degradation of oxidized cellulose;
[0019] FIG. 2 is a top view, side-by-side photo of an irradiated
oxidized cellulose implant according to the disclosure in a
hydrated state and a comparative non-irradiated oxidized cellulose
implant also in a hydrated state;
[0020] FIG. 3 is a graphical representation of degree of oxidation
for both an irradiated oxidized cellulose according to the present
disclosure and a non-irradiated oxidized cellulose;
[0021] FIG. 4 is a graphical representation of burst strength,
cellulose content and surface area for an irradiated oxidized
cellulose according to the disclosure;
[0022] FIG. 5 is a graphical representation of burst strength,
cellulose content and surface area for a non-irradiated oxidized
cellulose sample;
[0023] FIGS. 6A-6C are SEM images for samples of native cellulose,
non-radiated oxidized cellulose, and an irradiated oxidized
cellulose according to the present disclosure, respectively
[0024] FIGS. 7A-7C are XRD images for samples of native cellulose,
non-radiated oxidized cellulose, and an irradiated oxidized
cellulose according to the disclosure, respectively;
[0025] FIG. 8 is a graphical representation of a series of in vitro
degradation profiles for irradiated oxidized cellulose according to
the disclosure;
[0026] FIG. 9 is a graphical comparison of in vitro degradation
profiles of a non-radiated oxidized cellulose and an irradiated
oxidized cellulose according to the disclosure;
[0027] FIG. 10 is a graphical representation of molecular weight
distributions for a native cellulose sample, an irradiated oxidized
cellulose sample according to the disclosure, and a residual sample
of an irradiated oxidized cellulose according to the disclosure
after in vitro degradation testing;
[0028] FIG. 11 is a top view photo of four oxidized cellulose
samples subjected to different levels of radiation;
[0029] FIGS. 12A-F are photos of an irradiated oxidized cellulose
sample of the disclosure taken at various time periods during an in
vivo animal study;
[0030] FIG. 13 is a graphical representation of in vitro
degradation profiles for irradiated oxidized cellulose samples
according to the present disclosure that were used in the in vivo
study measured against a commercial oxidized cellulose sample of
the prior art.
DETAILED DESCRIPTION
[0031] In this document, the terms "a" or "an" are used to include
one or more than one and the term "or" is used to refer to a
nonexclusive "or" unless otherwise indicated. In addition, it is to
be understood that the phraseology or terminology employed herein,
and not otherwise defined, is for the purpose of description only
and not of limitation. Furthermore, all publications, patents, and
patent documents referred to in this document are incorporated by
reference herein in their entirety, as though individually
incorporated by reference. In the event of inconsistent usages
between this document and those documents so incorporated by
reference, the usage in the incorporated reference should be
considered supplementary to that of this document; for
irreconcilable inconsistencies, the usage in this document
controls. When a range of values is expressed, another embodiment
includes from the one particular value and/or to the other
particular value. Similarly, when values are expressed as
approximations, by use of the antecedent "about," it will be
understood that the particular value forms another embodiment. All
ranges are inclusive and combinable. Further, reference to values
stated in ranges includes each and every value within that range.
It is also to be appreciated that certain features of the invention
which are, for clarity, described herein in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention that are,
for brevity, described in the context of a single embodiment, may
also be provided separately or in any subcombination
[0032] As used herein, "body of cellulose" and derivations and
variations thereof, for example "cellulose body," "body of
irradiated cellulose," "body of oxidized cellulose," "body of
microbial cellulose," etc. is meant to describe a mass of cellulose
in any type of shape or spatial arrangement, and is not intended to
limit the mass of cellulose to any particular orientations or
configurations, unless otherwise explicitly stated herein.
Non-limiting examples of bodies of cellulose according to the
present disclosure can include a sheet of cellulose, a cellulose
membrane, a pellicle of cellulose, a cellulose film, a cellulose
patch and/or a cellulose sample.
[0033] As used herein "native cellulose", and derivations and
variations thereof, is meant to describe cellulose, both plant and
microbial originated forms, that are in an unadulterated state. For
example, in certain embodiments described herein "native cellulose"
refers to celluloses of any origin that have not been subjected to
any forms of oxidation or irradiation.
[0034] According to the present disclosure, a resorbable
biomaterial of irradiated oxidized cellulose is described that is
formed from a precursor reactive mixture of an irradiated cellulose
and an oxidizing agent. The reaction product thereof is a
resorbable biomaterial that is non-pyrogenic and can be porous.
Cellulose can be derived from either plant or microbial sources.
According to one embodiment, the irradiated cellulose is a
microbial-derived cellulose, and preferably is derived from
Gluconacetobacter xylinus.
[0035] Any suitable oxidizing agent can be used in the reactive
mixture to react with the irradiated cellulose according to the
present disclosure. Some examples of suitable oxidizing agents can
include metaperiodate, hypochlorite, dichromate, peroxide,
permanganate or nitrogen dioxide. A preferred oxidizing agent is
sodium metaperiodate. The oxidizing agent can have, according to
one embodiment, a concentration range of about 0.01M to about
10.0M, preferably about 0.05M to about 1.0M, and more preferably
from about 0.1M to about 0.5M.
[0036] Irradiation of a cellulose body can effect changes in a
subsequent oxidation reaction by providing chemical, structural and
morphological changes within the cellulose body's fiber network.
For example, radiation treatment can, among other things, increase
cationic permselectivity, membrane conductivity, and cause
interhydrogen bonding changes. Radiating cellulose's chemical
structure of glucopyranose chains can decrease cellulose
crystallinity and average molecular weight, and increases the
available surface area. Without being bound by any particular
theory, it is believed that the chemical and physical changes of
the cellulose membrane that may result from treatment with
irradiation make it more amenable to chemical treatment; i.e.,
oxidation. It is also further believed that irradiation of the
cellulose membrane prior to oxidation results in a porous
biomaterial having shorter and more efficiently oxidized
glucopyranose chains which are more easily accessible by
biocompatible fluids. In contrast, it is expected that a
non-irradiated oxidized cellulose has, on average, longer
glucopyranose chains that contain more randomly scattered
dialdehyde groups and also continues to maintain a relatively high
crystalline structure. Shorter glucose chains formed from
irradiation can therefore result in a body of cellulose having a
greater overall amount of oxidized cellulose than can be achieved
in oxidation of a corresponding non-irradiated cellulose body. A
higher percentage of oxidized glucose chains can lead to a more
rapid and homogenous degradation of the irradiated oxidized
cellulose body. As previously noted, and depicted in FIG. 1, it is
hypothesized that in vivo degradation of oxidized cellulose occurs
primarily by hydrolysis into 2,4-dihydroxybutyric acid and glycolic
acid. Up to 90% of the degradation of the cellulose body can occur
in this manner. Once the degradation process is initiated it
continues along the glucose chains that comprise the cellulose
body. Additional degradation, which can account for the remaining
10% of the cellulose body, also occurs where hydrolysis of the
dialdehyde groups has fractured the large glucose chains into
smaller poly or oligosaccharide units which are further cleared
from the body through phagocytosis.
[0037] The non-pyrogenic resorbable biomaterial as described can
have a variable range of degree of oxidation, which can, according
to one embodiment, be in the range of about 0 percent to about 99
percent oxidation, for example in a range of about 20 percent to
about 70 percent. The degree of oxidation of the irradiated
oxidized cellulose can depend on the oxidizing agent selected, the
concentration range of the oxidizing agent, reaction temperature,
and the time period of the reaction between the irradiated
cellulose and the oxidizing agent. According to one embodiment, the
degree of oxidation is in the range of about 15 percent to about 80
percent, and in another embodiment is in the range of about 20 to
about 70 percent.
[0038] According to the present disclosure, an implant is described
having sufficient mechanical strength, conformability to anatomical
surfaces, and resorption profile for use in tissue repair,
replacement and/or augmentation procedures, particularly soft
tissue applications, and more particularly for use as a dural
replacement patch. The implant includes a porous body of irradiated
oxidized cellulose formed by reacting irradiated cellulose with an
oxidizing agent. The porous body of cellulose is non-pyrogenic and
has a heterogeneous three-dimensional fibrillar network of
cellulose that can transition from a first rigid (dehydrated) state
to a second hydrated state upon contact with biocompatible fluids
(e.g., water, saline, blood, CSF etc.). FIG. 2 is a top view of an
implant 10 according to one embodiment of the present disclosure in
a hydrated state and a non-irradiated oxidized cellulose implant 20
in a hydrated state. The implant 10 in the second hydrated state
can, according to one embodiment, be translucent, as shown in FIG.
2, and be in the form of a cellulose patch. As used herein
"translucent" refers to the ability of the implant, in a hydrated
state, to allow light to pass through in a diffused manner so that
a field is illuminated but objects cannot necessarily be seen
distinctly through the implant.
[0039] The porous characteristic of the implant both permits rapid
uptake of fluid (hydration) as well as allowing tissue ingrowth
when implanted. According to one embodiment, the implant in the
hydrated state has sufficient durability and burst strength
(explained in further detail below) to be manipulated and implanted
to a desired anatomical location and exhibits desired adherence and
attachment to both regular and irregular contoured anatomical
surfaces. According to one embodiment, a surface of the implant in
the hydrated state is conformable (explained in further detail
below) to an anatomical surface, preferably a surface of a soft
tissue and more preferably a dural tissue surface. The implant can,
in a further embodiment, adhere to an anatomical surface without
the aid of suturing or securing devices; i.e., the implant can be
self-adhering/self-securing. It should be appreciated, however,
that the implant can be secured to an anatomical surface with the
aid of suturing or securing devices if so desired.
[0040] In certain medical procedures, it is desirable to have
additional medical devices present at the anatomical location in
order to provide additional support, fixation and/or stabilization
at the locus of repair. Where such secondary medical devices are
desired, the implant surface in the hydrated state can be
conformable to the anatomical surface, the secondary medical device
surface, and/or both surfaces. Example of suitable secondary
medical devices can include, but are not limited to, bone screws,
bone plates, metallic and polymer meshes, as well as metallic and
polymer plates and caps such as those used in cranial
surgeries.
[0041] The porous body of irradiated oxidized cellulose that forms
the implant has the ability, according to one embodiment, to
transition from a first rigid (dehydrated) state to a second
hydrated state upon contact with a biocompatible fluid. An implant
in the second hydrated state has conformability to an anatomical
surface as is described below in further detail. In certain
embodiments, the transition can occur in a short time period. For
example, according to one embodiment, the implant can transition
from a first rigid state to a second hydrated state within about
less than 10 minutes. According to further embodiments, the implant
can transition from a first rigid state to a second hydrated state
(e.g., fully hydrated) within about less than five minutes, within
about less than 30 seconds, within about less than 10 seconds,
within about less than 5 seconds, or within about less than 2
seconds.
[0042] The porous body of irradiated oxidized cellulose that forms
the implant can further, according to some embodiments, hold and
retain a quantity (measured in either mass or volume) of
biocompatible fluid in the second hydrated state that is greater
than the dry mass of the implant in the first rigid state. The
amount of hydration that the implant can achieve in transitioning
from the first rigid state to the second hydrated state can be
measured by its Water Holding Capacity (WHC) value. The WHC value
will be explained in detail further below, but generally is a
measurement of the mass of the biocompatible fluid the implant in
its second hydrated state retains relative to the dry mass of the
implant in its first rigid state. The higher the WHC value is, the
greater the ability of the implant to take up biocompatible fluids.
Without being bound to any particular theory, it is believed that
the ability of the implant to take up a sufficiently large quantity
of fluid, relative to its dry weight size, can have a direct
correlation to the implant surface's ability to conform to both
regular and irregular anatomical surfaces and secondary medical
device surfaces. According to one embodiment, the implant has a WHC
of at least about 7.0, where the oxidizing agent has a
concentration of 0.3M or greater. According to another embodiment,
the ratio of the WHC value of the implant to its surface area
(measured in square centimeters) is at least about 2.7:1.
[0043] The implant has a variable range of degradation profiles
that can be manipulated to align with the clinical indication for
which it is intended to be implanted. For example, when the implant
is selected for use as a dural replacement patch, the porous body
that forms the implant can have a degradation profile that
substantially matches the natural tissue replacement rate of native
dura mater. In vitro degradation testing, done under conditions
simulating an in vivo environment, can be done to evaluate an
implant's degradation profile with respect to a desired clinical
indication, for example, as a dura replacement or a hemostat. In
vitro testing can be conducted for any length of time as is
desired, for example, one day, one week, four weeks, two months,
six months, one year, or multiple years. According to one
embodiment, the porous body has a one week in vitro degradation
profile (as explained in further detail below) under simulated body
fluid (SBF) conditions in the range of about zero to about 90
percent. According to another embodiment, the porous body has a one
week in vitro degradation profile in the range of about zero to 40
percent, when the oxidizing agent has a concentration of
approximately 0.1M. According to yet another embodiment, the porous
body has a one week in vitro degradation profile in the range of
about 20 to 90 percent, when the oxidizing agent has a
concentration of approximately 0.3M. According to still another
embodiment, the porous body has a one week in vitro degradation
profile in the range of about zero to 60 percent, when the porous
body has been oxidized for at least one hour. According to a
further embodiment, the porous body has a one week in vitro
degradation profile in the range of about 15 to 80 percent, when
the porous body has been oxidized for at least three hours. In
certain preferred embodiments the porous body has an in vitro
degradation rate, measured over four weeks, of about 80% to about
100%.
[0044] According to a further embodiment of the disclosure, the
implant can be a scaffold or carrier for one or more active agents.
The active agent or agents can be impregnated within the porous
body of cellulose that forms the implant, coated onto a surface of
the implant, and/or both. According to one embodiment, the active
agent or agents can be impregnated within and/or coated onto the
implant substantially at or near the time of implantation (i.e.,
intraoperatively). In an alternative embodiment, the active agent
or agents can be impregnated within and/or coated onto the implant
prior to the time of implantation (i.e., preoperatively). In
certain embodiments, more than one active agent can be impregnated
within and/or coated onto the implant, and further the more than
one active agents can be impregnated within and/or coated onto the
implant at different time periods. For example, some active agents
can be preoperatively combined with the implant, while other active
agents can be combined intraoperatively. Active agents that can be
utilized with the implant include any compositions suitable for
treatment at the anatomical location, such as, bone marrow,
autograft, osteoinductive small molecules, osteogenic material,
stem cells, bone morphogenic proteins, antibacterial agents,
calcium phosphate ceramics, and mixtures and blends thereof.
[0045] The present disclosure further describes a method of
producing a body of oxidized cellulose that is porous and
resorbable including
[0046] (a) irradiating a body of cellulose so as to form an
irradiated body of cellulose, and
[0047] (b) reacting the irradiated body of cellulose with an
oxidizing agent so as to form a body of oxidized cellulose.
[0048] The body of oxidized cellulose formed can be, according to
one embodiment, porous, non-pyrogenic, and resorbable.
[0049] According to one embodiment the method can further include
the step of partially dehydrating the body of irradiated cellulose,
preferably by mechanically pressing the cellulose body. According
to another embodiment, the method can further include the step of
at least partially dehydrating the body of oxidized cellulose,
preferably by critical point drying using supercritical carbon
dioxide. According to a further embodiment, the method can include
contacting the non-pyrogenic body of cellulose, the irradiated body
of cellulose, and/or the body of oxidized cellulose with one or
more active agents.
[0050] Any suitable oxidizing agent can be used in reacting with
the irradiated body of cellulose according to the present method.
Some examples of suitable oxidizing agents can include
metaperiodate, hypochlorite, dichromate, peroxide, permanganate or
nitrogen dioxide. A preferred oxidizing agent is sodium
metaperiodate. According to one embodiment of the method, the
cellulose and metaperiodate react in a molar ratio range of 1:1 to
about 1:160 of cellulose to metaperiodate, and in another
embodiment, the cellulose and metaperiodate react in a molar ratio
range of 1:1 to about 1:120 of cellulose to metaperiodate. In a
preferred embodiment, the cellulose and metaperiodate react in a
molar ratio of about 1:120 of cellulose to metaperiodate. The molar
concentration range of the oxidizing agent can vary as desired.
According to one embodiment of the method, the oxidizing agent has
a concentration range of about 0.05M to about 1.0M in the reaction,
and in another embodiment, the oxidizing agent has a concentration
range of about 0.1M to about 0.4M in the reaction. Likewise the
reaction time between the irradiated body of cellulose and the
oxidizing agent can vary as desired. According to one embodiment of
the method, the oxidizing agent and the cellulose react for about
0.1 hours to about 72 hours, and in another embodiment, the
oxidizing agent and the cellulose react for about 3 hours to about
12 hours. For example, at or near a reaction temperature of
40.degree. C., the oxidizing agent can react with the cellulose at
a concentration and time range of about 0.1M for about 5 hours, to
about 0.5M for about 12 hours. Preferably, the oxidizing agent can
be present in a concentration range of about 0.2M to about 0.4M for
about 5 hours.
[0051] Reacting the irradiated body of cellulose with an oxidizing
agent to form a body of oxidized cellulose according to the methods
of the present disclosure can yield a variable degree of oxidation.
According to one embodiment of the method, the body of oxidized
cellulose has a degree of oxidation of at least about 25% after one
hour of reacting between the oxidizing agent and the cellulose.
According to another embodiment, the body of oxidized cellulose has
a degree of oxidation of at least about 40% after two hours of
reacting between the oxidizing agent and the cellulose. And in a
further embodiment, the body of oxidized cellulose has a degree of
oxidation of at least about 45% after two hours of reacting between
the oxidizing agent and the cellulose. In certain embodiments,
bodies of oxidized cellulose formed according to the embodiments of
the method described herein have a degree of oxidation in the range
of about 20% to about 70%.
[0052] According to one embodiment of the present disclosure a
method or methods of production can be utilized in the following
manner.
[0053] Preparation of the Cellulose Body
[0054] In preparing the resorbable biomaterial of the disclosure,
Gluconacetobacter xylinus (Acetobacter xylinum) cells are cultured
(incubated) in a bioreactor containing a liquid nutrient medium at
about 30.degree. C. at an initial pH of about 4.1-4.5. Cellulose
production can be achieved using, for example, sucrose as a carbon
source, ammonium salts as a nitrogen source, and corn steep liquor
as nutrient source. The fermentation process is typically carried
out in a shallow bioreactor with a lid which reduces evaporation.
Such systems are able to provide oxygen-limiting conditions that
help ensure formation of a uniform cellulose membrane. Dimensions
of the bioreactor can vary depending on the desired shape, size,
thickness and yield of the cellulose being synthesized.
[0055] The main fermentation process, following the incubation
step, is typically carried out under stationary conditions for a
period of about 8-120 hours, preferably 24-72 hours, during which
the bacteria in the culture medium synthesize and deposit thin
layers of cellulose sheets containing the microorganisms, thus
forming a cellulose membrane. Depending on the desired thickness
and/or cellulose yield, the fermentation can be stopped, at which
point the membrane can be harvested from the bioreactor. According
to one embodiment, the main fermentation is stopped after a
relatively short period to yield a uniform, low cellulose content
membrane (pellicle). The excess medium contained in the pellicle is
then removed by standard separation techniques such as compression
or centrifugation, which results in a partially dehydrated
pellicle.
[0056] Cellulose Body Purification
[0057] The partially dehydrated cellulose pellicle can then be
subject to a purification processing that renders the cellulose
nonpyrogenic. According to one embodiment the purification method
is a chemical purification of the cellulose membrane. The cellulose
is subjected to a series of caustic (e.g., concentrated sodium
hydroxide) chemical wash steps to convert the cellulose membrane
into a nonpyrogenic material, followed by soaking and/or rinsing
with filtered water, until a neutral pH is achieved. Alternatively,
or in conjunction with these steps, a short soak in diluted acetic
acid can also be conducted to ensure neutralization of the
remaining sodium hydroxide. Purification processes using various
exposure times, concentrations and temperatures, as well as
mechanical techniques including pressing, can be utilized on the
unpurified cellulose membrane. Processing times in sodium hydroxide
of about 1 to about 12 hours have been studied in conjunction with
temperature variations of about 30.degree. C. to about 100.degree.
C. to optimize the process. A preferred or recommended temperature
processing occurs at or near 70.degree. C.
[0058] The amount of endotoxins left in the cellulose body after
processing may be measured by Limulus Amebocyte Lysate (LAL) test.
The cleaning process described herein is capable of providing a
nonpyrogenic cellulose membrane (<0.06 EU/ml), which meets the
FDA requirements for dura substitute materials. Following the
purification of the cellulose membrane, according to one
embodiment, the pellicle can be mechanically compressed to a
desired weight and thickness.
[0059] Irradiation of the Cellulose Body
[0060] According to the disclosure, the non-pyrogenic cellulose
membrane is irradiated with ionizing radiation. According to one
embodiment, the radiation is .gamma.-radiation. The cellulose
membrane can absorb transmitted radiation in a range of about 10
kGy to about 100 kGy, and more preferably about 20 kGy to about 40
kGy. In a particular embodiment, the cellulose membrane can absorb
transmitted .gamma.-radiation in a range of about 20 kGy to about
26.5 kGy. In one embodiment of the disclosure the radiation is
provided in a single exposure or dosage. In an alternative
embodiment, the radiation can be provided through more than one
exposure. For example, the cellulose body according the disclosure
can be irradiated once, twice, or three times according to the
disclosure. Further, where more than one dosage or exposure is
applied to the cellulose body, the radiation transmitted and
absorbed by the cellulose body for each of the multiple dosages can
be of varying ranges. It should be appreciated by one skilled in
the art that the number of exposures and the intensity of the
radiation can be varied as desired.
[0061] In addition to irradiation, the cellulose membrane may be
presoaked in an electrolyte solution in order to promote a more
uniform oxidation and increase the rate of oxidation. The
electrolyte may be from the sulfate or chloride series, preferably
NaCl. The electrolyte concentration may be in the range from about
0.001M to about 1.0M, preferably about 0.05M to about 0.1M, and
more preferably about 0.2M to about 0.4M. The presoak may last in
the range of 30 minutes to 1 month, preferably 10 hours to 24
hours.
[0062] Oxidation of the Irradiated Cellulose Body
[0063] Following the irradiation and optional presoak steps, the
cellulose membrane is then reacted with a suitable oxidizing agent,
which could include, for example, chromic acid, hypochlorite,
dichromate, nitrogen dioxide, nitrogen tetroxide, or sodium
metaperiodate. According to one embodiment, the oxidizing agent is
sodium metaperiodate. It should be noted that when selecting
metaperiodate, the reaction is preferably conducted in the dark.
According to one embodiment, the oxidation reaction with the
oxidizing agent is for a time period in the range of about 30
minutes to 72 hours, preferably about 2-16 hours, and more
preferably about 2-6 hours. The oxidation reaction can typically
proceed at a temperature range of 18.degree. C. to 60.degree. C.,
preferably 30.degree. C. to 50.degree. C., and more preferably at
about 40.degree. C. According to another embodiment, the oxidation
reaction with the oxidizing agent is for a time period of at least
about one hour, and in yet another embodiment for at least about 3
hours. The container(s) are placed on a shaker and agitated at
20-500 rpm, preferably 350-450 rpm. The molar ratio between
cellulose and metaperiodate can be maintained at the range of
1:1-1:160, preferably 1:1-1:120, and more preferably at about
1:120. Upon completion of the oxidation reaction, the oxidized
cellulose membrane can be washed multiple times in filtered water
on an ice-bath to remove excess metaperiodate. Alternatively, it
can be washed in ethylene glycol to neutralize metaperiodate
followed by multiple rinses in DI water.
[0064] In an addition to, or alternatively to the oxidation process
previously described, prior to oxidation, the cellulose membrane
can be ground up to form a slurry and then homogenized into a fine
suspension of cellulose fibers. The homogenized suspension is then
oxidized with sodium metaperiodate as described previously. An
oxidized cellulose suspension is then recovered and washed to
remove the excess of metaperiodate. The suspension is then placed
in a mold and cross-linked to form a stable oxidized cellulose
membrane again.
[0065] In yet another alternative embodiment, the cellulose
membrane can undergo critical point drying prior to being oxidized.
Critical point drying is a stepwise process wherein water in the
cellulose membrane is exchanged with a non-aqueous solvent that is
soluble with water, for example ethanol. The ethanol is then
displaced with liquid carbon dioxide. This drying process can
enhance the penetration of the oxidizing agent into the cellulose
membrane. The dried membrane is reacted with the oxidizing agent,
as described above, and recovered and washed in a manner as
described above.
[0066] Drying of the Cellulose Body Using Supercritical Carbon
Dioxide
[0067] Following any of the oxidation processes described above,
the cellulose membrane can be further dried by critical point
drying utilizing supercritical carbon dioxide. As previously
explained above, the water in the cellulose membrane is exchanged
with a non-aqueous solvent (e.g., ethanol). The solvent is then
replaced with liquid carbon dioxide through a process called
critical point drying. During critical point drying, the cellulose
membranes are loaded onto a holder, sandwiched between stainless
steel mesh plates, and then soaked in a chamber containing
supercritical carbon dioxide under pressure. The holder is designed
to allow the CO.sub.2 to circulate through the cellulose membrane
while mesh plates stabilize the membrane to prevent the membrane
from waving during the drying process. Once all of the organic
solvent has been removed (which in most typical cases is in the
range of about 1-6 hours), the liquid CO.sub.2 temperature is
increased above the critical temperature for carbon dioxide so that
the CO.sub.2 forms a supercritical fluid/gas. Due to the fact that
no surface tension exists during such transition, the resulting
product is a dried membrane which maintains its shape, thickness
and 3-D nanostructure. The dried product undergoes cutting,
packaging and sterilization.
EXAMPLES
[0068] Unless otherwise stated herein, irradiated cellulose used in
the examples below was irradiated in a range of about 20-26.5
kGy.
[0069] Unless otherwise stated herein, native cellulose used in the
examples had a similar cellulose content (measured in g/cm.sup.2)
as the oxidized celluloses (both irradiated and non-irradiated)
prior to their undergoing either irradiation and/or oxidation.
[0070] Percent Oxidation of Samples
[0071] The percentage of oxidized cellulose in a cellulose membrane
was determined by measuring the amount of aldehyde content present.
For example, the oxidized samples were reacted with 10 ml 0.05M
NaOH at 70.degree. C. for 15-25 minutes in a stirred beaker. The
suspension was then cooled to room temperature and 10 ml of 0.05M
HCl was added to neutralize NaOH. The excess of acid was titrated
with 0.01M NaOH using phenolphthalein as an indicator. The
following formula was used to calculate the oxidation percentage of
the cellulose sample:
Oxidation %=[(M.sub.NaOH Tit*V.sub.NaOH Tit)*(MW.sub.oxidized
cellulose/M.sub.oxidized cellulose)*100]/2
TABLE-US-00001 TABLE 1 M.sub.NaOH Tit Molarity of NaOH used for
titration w/ phenolphthalein indicator V.sub.NaOH Tit Volume of
NaOH used in titration step MW.sub.oxidized cellulose Molecular
weight of oxidized cellulose (162 g/mol) M.sub.oxidized cellulose
Mass in grams of oxidized cellulose sample 100 Used to covert to
percentage 2 To account for dialdehyde nature of oxidized
cellulose
[0072] FIG. 3 is a graphical representation displaying the degree
of oxidation calculated according to the methodology described
above for both an irradiated oxidized cellulose according to the
present disclosure and a non-irradiated oxidized cellulose. Sodium
periodate was used as the oxidizing agent at a constant
concentration of 0.3M and constant temperature of 40.degree. C.
Percentage of oxidation was measured in samples over a time period
of 0-4 hours.
[0073] Conformability Testing
[0074] Conformability was tested by rehydrating dehydrated
cellulose samples in a solution of SBF (pH=7.4) and testing its
ability to conform to irregularities on the anatomically correct
surface of a cranial pulsation model (Synthes, Inc.). Dry oxidized
implant samples (both irradiated and non-radiated), oxidized at
0.3M periodate, 40.degree. C., 3 hrs, were placed on the moist
surface of the cranial pulsation model and rinsed with SBF. A
conformable sample was defined as: 1) displaying rapid rehydration
(transition from the first rigid state to second hydrated state),
for example, within 30 seconds, within 20 seconds, within 10
seconds, and preferably within 5 seconds; 2) complete attachment to
the surface of the model; and 3) adherence to the surface during
simulated pulsation for up to 1 minute.
[0075] The cranial pulsation model used is shown and described in
WD Losquadro et al., "Polylactide-co-glycolide Fiber-Reinforced
Calcium Phosphate Bone Cement," Arch Facial Plast Surg, 11(2),
March/April 2009, pp. 105-106. The pulsation model was designed and
manufactured by Synthes, Inc. The model consisted of 6 anatomically
correct adult skulls having various diameter openings that
simulated cranial defects. The skulls were made from solid foam
polyurethane and dura mater made from silicone. Each skull was
attached to an individual water pump with water sealed from the
external environment and the water from the pump capable of being
forced into the interior of the simulated dura mater material to
mimic dural pulsations.
[0076] To simulate a surgical wound environment, the skull model
was housed in a closed water bath maintained at a constant
37.degree. C. and 95%-100% relative humidity using a circulating
water heater. Water within the bath reached the base of the model
skulls but did not bathe the defect area. The closed water pump was
programmed to simulate intraoperative observations of dura
pulsation displacement of about 1.7 mm to 2.0 mm.
[0077] Burst Strength Testing
[0078] Oxidized cellulose samples of various sizes were tested for
ball burst strength using a manual burst tester, made by Synthes,
USA and calibrated at 11.4 kg (25 lbs.). The testing methodology
used to measure burst strength was based upon the procedures
described ASTM D2207-00 (Reapproved 2010), "Standard Test Method
for Bursting Strength of Leather by the Ball Method." The dry
samples are rehydrated in the SBF for 5 minutes and then sandwiched
in a stainless steel holder containing a central opening of 1 inch
diameter. The test method is designed to measure the bursting
strength of the sample by measuring the force required to force a
spherical ended plunger through the oxidized cellulose membrane;
that is, the plunger is used to penetrate the samples until failure
while force is measured digitally.
[0079] Cellulose Content Measurement
[0080] Samples with known surface area were air dried in the oven
at 55.degree. C. overnight. Cellulose content was measured by
dividing the weight of the dried sample by its surface area and was
expressed in g/cm.sup.2.
[0081] Data relating to the above experiments including cellulose
content, surface area, burst strength and conformability for an
irradiated oxidized cellulose sample and a non-irradiated oxidized
cellulose sample are graphically depicted in FIGS. 4 and 5,
respectively. The samples were oxidized with sodium metaperiodate
at a constant concentration of 0.3M at 40.degree. C. over a time
range of about 0-5 hours. The irradiated oxidized samples tested
and depicted in FIG. 4 were conformable when rehydrated at all
values as measured according to the standards as previously
described for conformability. In contrast, the non-irradiated
oxidized samples tested and depicted in FIG. 5 were conformable
when rehydrated only within values to the left of the dashed
vertical line, i.e., at an oxidation time of less than 2 hours.
[0082] SEM Observations
[0083] Samples of cellulose membranes including native cellulose,
non-irradiated oxidized cellulose and irradiated oxidized cellulose
were dried with supercritical CO.sub.2 and then coated with gold.
Oxidation was carried out at 0.3M periodate, 40.degree. C., 3 hrs.
A Hitachi field emission scanning electron microscope operating at
20 kV was used for examinations of the samples. FIGS. 6A-6C are SEM
images of samples of native cellulose, non-radiated oxidized
cellulose, and radiated oxidized cellulose samples, respectively.
The SEM images show that native cellulose, as shown in FIG. 6A have
a fibrillar, 3-dimensionally oriented and ordered structure of
cellulose chains. The non-radiated oxidized cellulose, as shown in
FIG. 6B, is a more compact structure than the native cellulose,
with regions of larger fibrils stacked together. The radiated
oxidized sample, as shown in FIG. 6C, is less ordered generally
than the previous cellulose samples, having a more chaotic
structure with generally smaller fibrils and generally higher
incidence of heterogenic regions than the other cellulose
samples.
[0084] X-ray Diffraction (XRD) Testing
[0085] Dried cellulose membrane samples, including native,
non-irradiated oxidized, and irradiated oxidized samples, were
placed in XRD sample cup holders, placed into the XRD magazine and
then into the device for measurement. Oxidation was carried out at
0.3M periodate, 40.degree. C., 3 hrs. X-ray diffraction spectra
were recorded using Ni filtered Cu-K.alpha. radiation produced by
the PANalytical XRD System. Scans were performed over the
4-90.degree. 2.theta. range, but analyzed from 4-40.degree.
2.theta. range. The data were analyzed with the HighScore Plus XRD
software. FIGS. 7A-7C are XRD spectrographs of the native,
non-irradiated oxidized, and irradiated oxidized samples,
respectively. As can be seen in the XRD displays, the native
sample, FIG. 7A, has a highly ordered crystalline structure,
followed by the non-radiated cellulose sample, FIG. 7B, with the
irradiated sample, FIG. 7C showing the least ordered crystalline
structure.
[0086] Percent crystallinity was calculated using the following
equation:
CrI=100.times.[(I.sub.002-I.sub.Amorph)/I.sub.002],
where CrI is the degree of crystallinity, I.sub.002 is the maximum
intensity of the (002) lattice diffraction (22.degree. 2.theta.)
and I.sub.Amorph is the intensity diffraction at 18.degree.
2.theta.. Table 2 below shows the measured crystallinity indexes
for the measured cellulose samples.
TABLE-US-00002 TABLE 2 Cellulose sample CrI [%] Native 81.9
Nonradiated oxidized 36.3 Irradiated oxidized 35.3
[0087] Rehydration/Water Holding Capacity Measurement
[0088] For this experiment, both irradiated and non-radiated bodies
of cellulose were cut into 4 cm.times.5 cm samples. These samples
were subjected to periodate solutions for oxidation at 40.degree.
C. and the following conditions:
TABLE-US-00003 Periodate Molarity (M) Time (Hours) 0.1 3 0.1 5 0.2
4 0.3 3 0.3 4 0.3 5 0.4 3 0.4 4 0.4 5 0.4 6
[0089] Reactions of both the irradiated and non-radiated cellulose
samples were done in duplicate. After each reaction was completed,
the samples were prepared for testing by washing and CO.sub.2
drying according to the methods previously disclosed herein.
[0090] Next, initial weight and surface area dimensions of all
samples were obtained, measuring non-radiated samples against their
irradiated counterpart. With a petri dish prepared with 20 ml SBF,
a non-radiated sample of cellulose was placed into the fluid for 30
seconds and then weighed out. This hydration step was then repeated
for the irradiated sample. Hydration for 30 seconds and then
weighing out wet mass was repeated for all samples prepared. The
water holding capacity (WHC) for each condition was calculated with
the following equation:
Wet mass ( g ) Dry Mass ( g ) = WHC ##EQU00001##
Averages of the WHC were taken to measure the difference between
non-radiated and irradiated cellulose in rehydration capabilities.
Also measured was the relationship between WHC and surface area
(SA) for each sample. Table 3 below shows the individual sample
results for each sample of radiated oxidized cellulose and
non-radiated oxidized cellulose at the given oxidation parameters.
Table 4 provides a summary of the average WHC and WHC/SA values for
each of the radiated and non-radiated oxidized samples at the given
oxidation parameters.
TABLE-US-00004 TABLE 3 Dry Mass Surface Area Wet Mass Average
WHC/Surface Avg. Sample Conditions (g) (cm2) (g) WHC WHC Area(SA)
(WHC/SA) 0.1M/3 Hrs Radiated 0.0357 13.02 1.0740 30.08 30.08 2.311
2.311 0.1M/3 Hrs Non-Radiated 0.0639 18.62 0.9750 15.26 15.26 0.819
0.819 0.1M/5 Hrs Radiated 0.0323 12.3 0.6970 21.58 21.58 1.754
1.754 0.1M/5 Hrs Non-Radiated 0.0643 13.26 0.9690 15.07 15.07 1.136
1.136 0.2M/4 Hrs Radiated 0.0281 6.16 0.4240 15.09 15.09 2.450
2.450 0.2M/4 Hrs Non-Radiated 0.0429 6.16 0.4580 10.68 10.68 1.733
1.733 0.3M/3 Hrs Radiated 0.0248 4.56 0.4040 16.29 14.12 3.572
3.085 0.3M/3 Hrs Radiated 0.0251 4.6 0.3000 11.95 2.598 0.3M/3 Hrs
Non-Radiated 0.0507 3.57 0.3470 6.84 7.02 1.917 1.965 0.3M/3 Hrs
Non-Radiated 0.0423 3.57 0.3040 7.19 2.013 0.3M/4 Hrs Radiated
0.0240 2.55 0.2350 9.79 9.06 3.840 3.382 0.3M/4 Hrs Radiated 0.0270
2.85 0.2250 8.33 2.924 0.3M/4 Hrs Non-Radiated 0.0604 2.52 0.2690
4.45 4.91 1.767 1.826 0.3M/4 Hrs Non-Radiated 0.0590 2.85 0.3170
5.37 1.885 0.3M/5 Hrs Radiated 0.0197 2.21 0.1660 8.43 8.69 3.813
4.058 0.3M/5 Hrs Radiated 0.0225 2.08 0.2014 8.95 4.303 0.3M/5 Hrs
Non-Radiated 0.0560 2.08 0.1990 3.55 3.49 1.708 1.732 0.3M/5 Hrs
Non-Radiated 0.0555 1.95 0.1900 3.42 1.756 0.4M/3 Hrs Radiated
0.0196 3 0.1830 9.34 9.62 3.112 3.207 0.4M/3 Hrs Radiated 0.0212 3
0.2100 9.91 3.302 0.4M/3 Hrs Non-Radiated 0.0577 2.38 0.2510 4.35
4.16 1.828 1.888 0.4M/3 Hrs Non-Radiated 0.0511 2.04 0.2030 3.97
1.947 0.4M/4 Hrs Radiated 0.015 1.8 0.1000 6.67 8.74 3.704 5.366
0.4M/4 Hrs Radiated 0.0175 1.54 0.1894 10.82 7.028 0.4M/4 Hrs
Non-Radiated 0.0453 1.95 0.1940 4.28 3.94 2.196 2.189 0.4M/4 Hrs
Non-Radiated 0.0425 1.65 0.1530 3.60 2.182 0.4M/5 Hrs Radiated
0.0170 1.3 0.1230 7.24 7.69 5.566 5.497 0.4M/5 Hrs Radiated 0.0140
1.5 0.1140 8.14 5.429 0.4M/5 Hrs Non-Radiated 0.0326 1.3 0.1100
3.37 3.24 2.596 2.476 0.4M/5 Hrs Non-Radiated 0.0360 1.32 0.1120
3.11 2.357 0.4M/6 Hrs Radiated 0.0112 1.2 0.1160 10.36 9.49 8.631
7.904 0.4M/6 Hrs Radiated 0.0137 1.2 0.1180 8.61 7.178 0.4M/6 Hrs
Non-Radiated 0.0418 0.9 0.1170 2.80 2.52 3.110 2.577 0.4M/6 Hrs
Non-Radiated 0.0307 1.1 0.0690 2.25 2.043
TABLE-US-00005 TABLE 4 Avg. WHC Avg. (WHC/SA) Oxid. Values Radiated
Non-Radiated Radiated Non-Radiated 0.1M/3 Hrs 30.08 15.26 2.3106
0.8195 0.1M/5 Hrs 21.58 15.07 1.7544 1.1365 0.2M/4 Hrs 15.09 10.68
2.4495 1.7331 0.3M/3 Hrs 14.12 7.02 3.0854 1.9651 0.3M/4 Hrs 9.06
4.91 3.3819 1.8263 0.3M/5 Hrs 8.69 3.49 4.0581 1.7320 0.4M/3 Hrs
9.62 4.16 3.2071 1.8876 0.4M/4 Hrs 8.74 3.94 5.3658 2.1890 0.4M/5
Hrs 7.69 3.24 5.4971 2.4762 0.4M/6 Hrs 9.49 2.52 7.9043 2.5766
[0091] In Vitro Degradation Profile
[0092] Samples of both irradiated and non-irradiated oxidized
cellulose having various degrees of oxidations, prepared according
to the disclosure, were tested in vitro by incubation in SBF.
Degradation profiles showed that the cellulose samples remained
mechanically stable (in the form of a membrane/film) over at least
a 2-4 week period. After that initial period, the samples began to
disintegrate into irregular cellulosic masses and degrade over the
following 1-3 months, leaving approximately 0.1%-5.0% of their
initial dry mass.
[0093] Both real-time and accelerated studies were conducted.
Samples of dried irradiated oxidized cellulose (approximately
1.times.1 cm squares) were placed in the sterile 50 ml centrifuge
conical tubes filled with 20 ml of SBF (pH=7.4) and kept in static
conditions at 37.degree. C. or 55.degree. C. for a period of time
between 1 week and 6 months (real time). For the real-time study,
the SBF in each tube was changed daily for 5 initial days and then
weekly by centrifuging samples, decanting old SBF and replacing it
with a fresh one. Samples were analyzed at 1, 2, 3, 4, 14, 28, 90
and 164 days. At each time point, tubes were centrifuged to collect
the residual pellet. The supernatant was decanted and DI water was
added to wash the pellet from residual SBF. The tubes were stirred
briefly and centrifuged again to collect pellet. The DI water
washing step was repeated twice. The pellet was then dried in the
oven at 60.degree. C. to constant weight. The percent of
degradation was calculated as difference between the dry pellet
weight and original sample weight.
[0094] FIG. 8 graphically shows degradation profiles (SBF; pH=7.4,
55.degree. C., 7 days) of irradiated cellulose, oxidized at
different periodate concentrations. For all conditions tested, a
progressive loss of the samples' dry mass was observed throughout
the study. Once incubated in SBF, samples become softer, gel-like
structures with a high degree of translucency. Depending on the
oxidation conditions used, a degradation range of about 10-95% can
be obtained after 7 days incubation time. The results show that
degradation rate is related to oxidation degree, which can be
controlled by periodate concentration, reaction temperature and
reaction time. A conformable and mechanically stable biomaterial
with desired degradation rate can be prepared by using such
approach. FIG. 9 graphically depicts the results of the in vitro
degradation (dry mass loss) for both irradiated and non-irradiated
cellulose samples, oxidized for various periods of time (1-4
hours). The curves show that there was a weight loss in both types
of samples oxidized for 3 and 4 hours. The initial rate of mass
loss for samples oxidized for less than about 3 hours is greater?
for the irradiated cellulose than the non-irradiated cellulose.
[0095] Samples of cellulose of the type used in the in vitro
degradation were submitted to Polymer Solutions Incorporated (PSI)
(Blacksburg, Va.) for analysis of molecular weight distributions
using GPC with light scattering detection. Three types of samples
were submitted: 1) a sample of native microbial cellulose,
identified as "Native Cellulose (wet);" 2) a sample of irradiated
oxidized microbial cellulose, identified as "Oxidized Cellulose
(wet);" and 3) a residual sample of irradiated oxidized microbial
cellulose that had been subjected to a seven day in vitro
degradation process as described above, identified as "Implant
Residual Content."
[0096] As used in this experiment, the term "wet" is used to
indicate that the "Native Cellulose" sample and the "Oxidized
Cellulose" sample did not undergo the step of critical point drying
with supercritical CO.sub.2 that was previously described. Both the
"Oxidized Sample" and the "Implant Residual Content" sample were
oxidized at 0.3M periodate, 40.degree. C., 3 hrs.
[0097] The molecular weight distributions of the cellulose samples
were analyzed using gel permeation chromatography (GPC) with light
scattering detection. Approximately half of the 4.times.5 cm piece
of Native Cellulose (wet) and the entire 2.2.times.3.0 cm piece of
Oxidized Cellulose (wet) were placed in separate 40-mL glass
scintillation vials. A piece of Whatman #1 filter paper was ground
for about 5 minutes in a small blade-type coffee mill, and
approximately 20 mg of the resulting "fluff" was weighed into a
40-mL scintillation vial. The Whatman filter paper was included as
a control for the dissolution process, and also for use in
estimating the specific refractive index increment (dn/dc) of
cellulose in DMAc. 10 mL of pure water and a disposable stir bar
were added to each vial. Each vial was stirred for approximately 5
hours at 50.degree. C. The Native Cellulose (wet) and Oxidized
Cellulose (wet) samples did not disintegrate. Therefore, the wet
cellulose pieces were placed in a small food processor with 60 to
70 mL of pure water and processed for 60 to 90 seconds, resulting
in slurries of very small fibrous particles. The slurries were then
vacuum filtered on 47-mm 0.2-.mu.m nylon membranes, just until
excess water was removed.
[0098] The wet cellulose samples were then transferred to Whatman
Vecta-Spin centrifuge filters, which contained 10-.mu.m
polypropylene mesh filters. The water was centrifuged off and
replaced with HPLC grade methanol and soaked overnight. The
following day the methanol was spun off, and an additional 3-hour
soak with fresh methanol was performed, followed by a 20-minute
centrifugation. The solvent exchange process was then repeated
using dried N,N-dimethylacetamide (DMAc) for 3 exchanges with soak
times of 75 minutes, overnight, and 30 minutes, with 20 minutes
centrifugation after each soak.
[0099] The DMAc-wet samples and Whatman filter paper control were
then transferred into 40-mL scintillation vials. 20 mg of the
Implant Residual Content sample was weighed into a 40-mL
scintillation vial as well. To each of these, 2 mL of a solution of
8% lithium chloride in DMAc and a stir bar were added. The samples
were stirred for 3 days at room temperature, and were then placed
in a refrigerator at 4.degree. C. for three additional days. The
Native Cellulose and the Whatman filter paper control were
completely dissolved. The Oxidized Cellulose sample formed a cloudy
solution with numerous gel-like particles. The Implant Residual
Content sample was mostly dissolved, but with a very small
percentage of the original sample that would not dissolve.
[0100] The Native Cellulose and Oxidized Cellulose solutions were
diluted with 14 mL of DMAc. The Whatman cellulose control and the
Implant Residual Content sample were diluted with 30 mL of DMAc.
The diluted solutions were stored at approximately 4.degree. C. for
an additional day before being filtered through 0.45-.mu.m pore
size PTFE syringe filters into GPC autosampler vials. Following
filtration, duplicate GPC injections of each sample solution were
performed under parameters listed in Table 5 below and molecular
weights were calculated using dual-angle light scattering.
TABLE-US-00006 TABLE 5 Parameter Value Mobile Phase: 0.5% LiCL in
DMAc Columns: (2) Tosoh Alpha M, 300 .times. 7.8 mm Flow Rate: 0.8
mL/min Column Temperature: 50.degree. C. Detectors: Visotek Triple
Detector Array (TDA) w/RI, 7.degree. and 90.degree. light
scattering, & differential viscometer detectors Dectector
Temperature: 50.degree. C. Injection Volume: 200 .mu.L Mol. Wt.
Calculation Dual-Angle Light Scattering Method: Light Scattering/RI
670 nm Detection Wavelength: dn/dc of cellulose: 0.1309 mL/g*
*dn/dc of cellulose value refers to value of cotton cellulose used
as control value
[0101] The molecular weight averages (Mn, Mw, Mz) and
polydispersity (Mw/Mn) are presented for duplicate injections of
each sample in Table 6. The molecular weight distribution plots of
all samples are compared and graphically depicted in FIG. 10. The
specific refractive index increment (dn/dc) value of cellulose in
DMAc, used for the light scattering molecular weight calculations,
was estimated from the RI detector peak area for duplicate
injections of the Whatman filter paper control. The bacterial
cellulose samples were assumed to have the same do/dc value as the
Whatman filter paper (cotton cellulose).
TABLE-US-00007 TABLE 6 M.sub.n M.sub.w Sample GPC Run (g/mol)
(g/mol) M.sub.z (g/mol) M.sub.w/M.sub.n Native 1 27,047 87,951
187,092 3.25 Cellulose 2 26,383 88,598 194,792 3.36 (wet) Average
26,715 88,275 190,942 3.30 Std. Dev. 470 457 5,445 0.08 Oxid. 1
22,598 75,899 216,679 3.36 Cellulose 2 22,633 76,687 248,783 3.39
(wet) Average 22,616 76,293 232,731 3.37 Std. Dev. 25 557 22,701
0.02 Implant 1 17,134 43,602 89,334 2.54 Residual 2 21,227 46,578
93,085 2.19 Content Average 19,181 45,090 91,210 2.37 Std. Dev.
2,894 2,104 2,652 0.25 Whatman #1 1 289,819 476,627 678,373 1.64
Filter Paper 2 295,673 472,848 680,254 1.60 (Control) Average
292,746 474,738 679,314 1.62 Std. Dev. 4,139 2,672 1,330 0.03
[0102] Radiation Dosage and In Vitro Degradation
[0103] Four cellulose bodies were subjected to varying radiation
dosages and then oxidized at 0.3M periodate, 40.degree. C., 3 hrs.
After undergoing oxidation, the samples' in vitro degradation rate
(7 days) was measured.
[0104] The cellulose bodies were sent to Sterigenics (Charlotte,
N.C.) to undergo radiation exposure at various dosages. The samples
were irradiated with gamma radiation using the ExCell.RTM. system,
a high-precision, low-volume irradiator. Each exposure of radiation
was intended to irradiate the samples in the range of about 20 kGy
to about 26.5 kGy. Actual dosage levels for each treatment were
measured to be about 23 kGy. Afterwards, the samples were oxidized
using 0.3M periodate at 40.degree. C. for three hours. FIG. 11 is a
top view of the four samples after radiation exposure and
subsequent oxidation. Sample 1, 41, was not radiated. Sample 2, 42,
was exposed to one treatment at a dose of 23 kGy. Sample 3, 43, was
exposed to two separate treatments, each at a dose of 23 kGy.
Sample 4, 44, was exposed to three separate treatments, each at a
dose of 23 kGy. The samples were measured for in vitro degradation,
as previously described, for one week at SBF conditions at
55.degree. C. Table 7 shows the measured percent of sample
degradation of each sample after one week at SBF conditions, along
with the sample weight, surface area, and cellulose content, prior
to the start of the in vitro degradation test.
TABLE-US-00008 TABLE 7 Cellulose Degradation Surface Area Content
Sample (7 days) Weight (g) (cm.sup.2) (10.sup.3 g/cm.sup.2)
Non-radiated 71% 0.0522 2.7 19.3 Single radiated 71% 0.0629 3.6
17.0 Double radiated 70% 0.0222 5.5 4.0 Triple radiated 71% 0.0202
7.2 2.8
[0105] While increased radiation may affect the dry weight and size
of the samples after oxidation, as shown in FIG. 11, there is not a
corresponding change in overall degradation, as can been seen in
Table 7. Without being bound by any particular theory, it is
believed that radiation likely caused two things to occur in the
tested samples: (1) chain scission occurred due to the formation of
free radicals, which lowered the average molecular weight of the
cellulose and (2) the free radicals promoted cross linking in the
cellulose structure. Therefore, while chain scission is likely the
dominant mechanism, the formation of small cross-linked molecules
of varying geometries is also likely, which may prevent further
degradation from occurring.
[0106] Again, without being bound by any particular theory, it is
believed that the lowering of the molecular weight of the cellulose
samples from increased exposure to radiation can cause an increased
size of the oxidized cellulose samples as shown in FIG. 11.
Further, any chain scission that occurs as a result of radiation
decreases the length of cellulose chains, which prevents the sample
from shrinking during oxidation. Non-radiated cellulose samples
with longer chain lengths are likely affected by the oxidization
procedure.
[0107] In Vivo Studies
[0108] The in vivo study evaluated in vivo degradation rate and
safety/biocompatibility of four irradiated oxidized cellulose
implants according to the present disclosure (identified as TD 1-TD
4), each having a different oxidation profile, and compared them to
1) a commercially available cross-linked bovine tendon collagen,
identified as CD 1, and 2) a native microbial cellulose, identified
as CD 2. The oxidation profiles of the four implants according to
the disclosure were as follows: TD 1 having a 55% oxidation
profile, oxidized at 0.4M periodate, 40.degree. C., 3 hrs.; TD 2
having a 84% oxidation profile, oxidized at 0.4M periodate,
40.degree. C., 4 hrs.; TD 3 having a 50% oxidation profile,
oxidized at 0.3M periodate, 40.degree. C., 3 hrs.; and TD 4 having
a 94% oxidation profile, oxidized at 0.3M periodate, 40.degree. C.,
5 hrs. All TD samples used in the in vivo studies were irradiated
prior to oxidation according to the process described herein.
[0109] Seventeen male New Zealand White rabbits, (16 study animals
plus 1 spare, per study protocol) were entered into the study. The
16 study animals were assigned to one of four groups of four
animals each. The implants were all implanted by subcutaneous
implantation in a rabbit model and evaluated at 2, 4, 12 and 26
weeks after implantation. Each animal received one of each of the
six materials, implanted into separate subcutaneous pockets on the
rabbit's back (three on each side of the dorsal midline). The
location of each different implant in each rabbit was randomized
according to a predetermined implantation matrix. The superficial
fascia was bluntly dissected away from the underlying tissue to
create a subcutaneous pocket deep enough to contain the test or
control device (native microbial cellulose and resorbable
collagen). After each test device or control device had been
positioned, a pair of small skin staples were used to mark the
location of the device and placed at the two corners of the test or
control device closest to the incision site, but not associated
with the material. A pair of 4-0 Prolene sutures was used to tack
down the implant to the underlying subcutaneous tissue in order to
prevent implant migration after implantation.
[0110] Four rabbits were euthanized and subjected to a limited
necropsy at each of four different time points: 2 weeks, 4 weeks,
12 weeks or 26 weeks after implantation surgery. Necropsy was
limited to gross observations of the implantation sites and
peri-implant tissues, with limited tissue collection (consisting of
collection from the operative sites of the implant surrounded by
peri-implant tissues). The degradation of the implants at each site
for each measurement period (2 weeks, 4 weeks, 12 weeks or 26
weeks) was recorded and is shown below in Tables 8-12,
respectively.
TABLE-US-00009 TABLE 8 Week 2 TD1 TD2 TD3 TD4 CD1 CD2 Terminal
Assessment .sup.a 2 weeks 2 weeks 2 weeks 2 weeks 2 weeks 2 weeks
Inflammation .sup.b 0.75 1.25 0.75 0.50 0.00 0.00 Infection .sup.b
0.00 0.00 0.00 0.00 0.00 0.00 Fibrosis .sup.b 0.00 0.00 0.00 0.25
0.00 0.00 Seroma .sup.b 0.00 0.25 0.00 0.00 0.00 0.00 Hematoma
.sup.b 0.00 0.00 0.00 0.00 0.00 0.00 Gross Vascularization .sup.b
1.25 1.00 0.50 0.75 0.00 0.00 Presence of Implant .sup.c 1.00 1.00
1.00 1.00 0.25 0.00 Implant Degradation .sup.d 2.50 2.50 2.50 2.75
0.25 0.00 Implant Measurement.sup.e 27.0 65.3 25.0 47.0 91.8 145.0
% of Original Implant 17.3 41.8 25.0 37.6 58.7 92.8 Area
Remaining
TABLE-US-00010 TABLE 9 Week 4 TD1 TD2 TD3 TD4 CD1 CD2 Terminal
Assessment .sup.a 4 weeks 4 weeks 4 weeks 4 weeks 4 weeks 4 weeks
Inflammation .sup.b 1.25 1.25 1.25 1.75 0.00 0.00 Infection .sup.b
0.00 0.00 0.00 0.00 0.00 0.00 Fibrosis .sup.b 0.00 0.00 0.00 0.00
0.00 0.00 Seroma .sup.b 0.00 0.00 0.00 0.00 0.00 0.00 Hematoma
.sup.b 0.00 0.25 0.25 0.25 0.25 0.00 Gross Vascularization .sup.b
0.75 0.25 0.50 0.50 0.00 0.00 Presence of Implant .sup.c 1.00 1.00
1.00 1.00 0.50 0.00 Implant Degradation .sup.d 2.50 2.75 2.50 2.75
0.75 0.00 Implant Measurement.sup.e 15.5 17.8 11.0 29.5 49.5 94.0 %
of Original Implant 9.9 11.4 11.0 23.6 31.7 60.1 Area Remaining
TABLE-US-00011 TABLE 10 Week 12 TD1 TD2 TD3 TD4 CD1 CD2 Terminal
Assessment .sup.a 12 Weeks 12 Weeks 12 Weeks 12 Weeks 12 Weeks 12
Weeks Inflammation .sup.b 1.00 0.75 0.75 0.50 0.00 0.50 Infection
.sup.b 0.00 0.00 0.00 0.00 0.00 0.00 Fibrosis .sup.b 0.00 0.00 0.25
0.00 0.00 0.50 Seroma .sup.b 0.00 0.00 0.00 0.00 0.00 0.00 Hematoma
.sup.b 0.00 0.00 0.00 0.00 0.00 0.00 Gross Vascularization .sup.b
0.50 0.75 1.25 0.75 0.25 1.25 Presence of Implant .sup.c 1.00 1.00
1.00 1.00 1.75 0.00 Implant Degradation .sup.d 3.00 2.75 3.00 3.00
3.50 0.00 Implant Measurement.sup.e 7.0 47.3 30.0 42.0 9.5 120.8 %
of Original Implant 4.5 30.2 30.0 33.6 6.1 77.3 Area Remaining
TABLE-US-00012 TABLE 11 Week 26 TD1 TD2 TD3 TD4 CD1 CD2 Terminal
Assessment .sup.a 26 Weeks 26 Weeks 26 Weeks 26 Weeks 26 Weeks 26
Weeks Inflammation .sup.b 0.00 0.00 0.00 0.00 0.00 0.00 Infection
.sup.b 0.00 0.00 0.00 0.00 0.00 0.00 Fibrosis .sup.b 0.25 0.25 0.38
0.25 0.00 0.38 Seroma .sup.b 0.00 0.00 0.00 0.00 0.00 0.00 Hematoma
.sup.b 0.00 0.00 0.00 0.00 0.00 0.00 Gross Vascularization .sup.b
1.00 0.75 0.88 1.00 0.00 1.00 Presence of Implant .sup.c 1.25 1.25
1.25 1.50 2.00 1.00 Implant Degradation .sup.d 3.25 3.25 3.25 3.50
4.00 1.00 Implant Measurement.sup.e 5.8 19.0 22.5 0.0 0.0 53.5 % of
Original Implant 3.7 12.2 22.5 0.0 0.0 34.2 Area Remaining
[0111] .sup.a Average score. [0112] .sup.b Scoring: 0=None;
1=Slight; 2=Moderate; 3=Severe [0113] .sup.c Scoring: 0=Material
present as implanted; 1=Material present, but signs of degradation;
2=Material not present [0114] .sup.d Scoring: 0=Same as when
implanted; 1=Slight fragmentation; 2=Moderate fragmentation;
3=Severe fragmentation; 4=Not able to score [0115] .sup.e Implant
measurement calculated in square millimeters (mm.sup.2)
[0116] The control implants did not show any inflammation of note.
After two weeks, there was some gross inflammation noted around all
test material implants, with the least amount being around TD4.
Inflammation increased slightly at all test material sites after
four weeks, with the most inflammation being observed around TD4.
At 12 weeks, inflammation at all sites was similar to that observed
at two weeks, with the least amount observed around TD4.
Inflammation was not observed around any of the implants at 26
weeks. No infection was observed at any time point. The TD2 implant
site in one animal was noted to have a possible infection, but when
examined microscopically, there was no evidence of infection, or
evidence of bacterial colonies. There was little to no fibrosis
observed grossly around any of the implants, except perhaps around
the native microbial cellulose implant after 12 weeks. At 26 weeks,
slight fibrosis was observed around all implants except at the
cross-linked bovine tendon collagen sites because it was not
present. There appeared to be a small seroma around the TD2 implant
site in one animal at two weeks. No other sites at any time point
contained a seroma.
[0117] One animal had evidence of a possible resolving hematoma
near the TD2 implant site, and two animals had evidence of possible
resolving hematomas associated with the TD4 implants, all after two
weeks. These were likely caused by the surgical procedure itself.
After four weeks, small hematomas were present in the CD1 implant
site in one rabbit, at the TD2 site in another animal, at the TD3
site in one animal and at the TD4 site in yet another animal. In
all cases, these were likely a result of placement of the stay
sutures. No hematomas were observed at either 12 or 26 weeks.
[0118] Gross vascularization (a sign of chronic inflammation) was
also rarely observed at the early time points, but tended to
increase at the 12 and 26-week time points, being greatest at the
latter. It was most prominent around the TD1 implant and least
prominent around the TD3 implant at 2 weeks. No gross
vascularization was observed around the control implants at 2 and 4
weeks, but it was evident after 12 weeks, especially around the
native microbial cellulose implant. The cross-linked bovine tendon
collagen and all test material implant sites also showed some gross
vascularization after 12 weeks. It was about equally present at all
sites, except the cross-linked bovine tendon collagen sites, where
it was not present at all at 26 weeks.
[0119] The representative necropsy images of test material TD1 are
shown in FIGS. 12A-F. FIG. 12A shows an embodiment of the implant
in a first rigid state immediately after placement in position in
subcutaneous pocket. After placement, the implant rapidly
transitioned to the second hydrated state by absorbing moisture
from the surrounding tissue, and subsequently conformed and adhered
to the tissue surface as shown in FIG. 12B. It should be noted that
after hydration the implant displayed translucency and is nearly
indistinguishable from the underlying tissue. FIG. 12C shows the
implant 2 weeks after implantation where the implant was measurably
thinner. FIG. 12D shows the implant 4 weeks after implantation
where the implant was moderately degraded with one comparatively
large piece remaining. FIG. 12E shows the implant 12 weeks after
implantation where the implant was severely degraded, there was
discoloration of the tissue, and the portion of the degraded
implant remaining was very diffuse and thin. FIG. 12F shows the
implant 26 weeks after implantation where the implant was severely
degraded; the portion of implant remaining was very diffuse and
thin. Stay sutures are visible, and arrows indicate diffuse small
areas of discoloration that may indicate fragments of remaining TD1
implant material.
[0120] The native microbial cellulose implant showed no sign of
degradation over the entire period of study. Cross-linked bovine
tendon collagen on the other hand, showed some degradation at 2
weeks, was significantly degraded at 4 weeks, and was essentially
not present at 12 and 26 weeks. All of the test devices showed
marked degradation at all time points, but interestingly, while
they initially appeared to degrade quickly, they did not continue
to degrade as rapidly. The in vivo study showed that at two weeks,
it appeared that TD1 and TD3 showed the most rapid degradation.
After four weeks, degradation of TD1, TD2 and TD3 were similar,
while TD4 showed less degradation. At 12 weeks, degradation of TD2,
TD3 and TD4 were similar, while TD1 appeared considerably more
degraded than any of the other test devices. At 26 weeks, no
cross-linked bovine tendon collagen was present, there were some
remnants of all test devices still present (in the form of tissue
discoloration), and the native microbial cellulose was still
present as implanted.
[0121] A similar behavior was observed for the samples (TD1-TD4)
previously tested in the in vivo study above, during an accelerated
in vitro degradation study. The in vitro study of TD1-TD4 against a
control sample of Johnson & Johnson Surgicel.RTM., showed a
very rapid initial degradation of irradiated oxidized cellulose
samples over the first 48 hours of incubation in SBF (pH=7.4) at
55.degree. C. FIG. 13 graphically depicts the degradation results
of the in vitro study. The study showed that for TD1-TD4, this
rapid degradation levels off at 72-96 hours achieving a
plateau.
[0122] Collected tissue samples from the in vivo implant sites were
fixed in 10% neutral buffered formalin (NBF) and sections through
the approximate center of the implant site were taken and embedded
in paraffin. Hematoxylin and eosin (H & E) staining and Schiff
staining (PAS) were performed. PAS staining was used to evaluate
aldehyde (oxidized cellulose) presence. All slides were examined
and reviewed by two board certified veterinary pathologists.
Evaluation of the tissue response to the test and control devices,
including scoring the degree of vascularization, fibrosis, and
immune response, of the test and control devices and scoring the
degree of irritation of the tissue at the implant site were
performed, following the ISO 10993 (2007), part 6, annex E
guidelines for evaluation of local biological effects after
implantation.
[0123] Microscopic evaluation revealed that TD1 and TD4
demonstrated notable loss of material apparent by 12 weeks, and
this was comparable in degradation to the cross-linked bovine
tendon collagen. TD2 and TD3 had delayed loss of implant, with
notable loss not occurring until the 26-week time point. The native
microbial cellulose implant showed little to no sign of degradation
over the entire period of study.
[0124] The inflammatory response to the implant materials was
consistent with a foreign body response, characterized by variable
numbers of macrophages, foreign body giant cells and with minimal
to mild numbers (a score of 1 to 2) of neutrophils. Eosinophils
were not uncommon and plasma cells were rarely seen. Fibrosis
generally consisted of narrow to moderately thick bands, with the
exception of the native microbial cellulose, which presented with
increased fibrous capsule formation around the implant at 12 weeks.
The total irritancy score was calculated from the sum of the
overall inflammatory response (times two), vascularity, and
fibrosis pathology scores. The total irritancy score was used to
determine the following severity grade for irritant status: [0125]
Non-irritant (0.0 to 2.9) [0126] Slight irritant (3.0 to 8.9)
[0127] Moderate irritant (9.0 to 15.0) [0128] Severe irritant
(>15.0)
[0129] Average ranked irritation scores were calculated for each
test device at each time point by subtracting the average irritancy
score for either CD1 or CD2 from each test device, and were based
upon the guidelines as described in ISO 10993, part 6, Annex E
(informative) "Examples of evaluation of local biological effects
after implantation" for scoring of histology. Tables 12 and 13,
below, show the average irritancy scores for the samples against
each of control CD1 and CD2 respectively.
[0130] The inflammatory reaction to TD4 (including the numbers of
macrophages and giant cells) was most prominent at the early time
points of all the test materials. These findings are consistent
with a very rapidly absorbed material. At 12 and 26 weeks,
macrophages and giant cells again predominated for all the test
materials, but the highest scores were seen in proximity to TD2,
and to a lesser extent, TD3. This finding likely indicates that
these materials were resorbing more slowly than was the TD4
material.
[0131] The four test materials were compared to the control
implants (native microbial cellulose and cross-linked bovine tendon
collagen) and were considered to be either non-irritants or slight
irritants at 2, 12 or 26 weeks. At the 4-week time point only, TD1
and TD4 were considered to be moderate irritants when compared to
the native microbial cellulose.
TABLE-US-00013 TABLE 12 TD1-TD4 against CD1 TD1 TD2 TD3 TD4 2 weeks
2.75 0.25 4.50 4.75 4 weeks 2.75 0.50 0.00 6.50 12 weeks 0.75 0.00
0.50 0.00 26 weeks 2.75 8.50 3.50 1.50
TABLE-US-00014 TABLE 13 TD1-TD4 against CD2 TD1 TD2 TD3 TD4 2 weeks
0.25 0.00 2.00 2.25 4 weeks 8.50 6.25 5.50 12.25 12 weeks 5.00 2.25
4.75 3.75 26 weeks 0.00 3.33 0.00 0.00
[0132] Although the present disclosure has been described in
accordance with several embodiments, it should be understood that
various changes, substitutions, and alterations can be made herein
without departing from the spirit and scope of the present
disclosure, for instance as indicated by the appended claims. Thus,
it should be appreciated that the scope of the present disclosure
is not intended to be limited to the particular embodiments of the
process, manufacture, composition of matter, methods and steps
described herein. For instance, the various features as described
above in accordance with one embodiment can be incorporated into
the other embodiments unless indicated otherwise. Furthermore, as
one of ordinary skill in the art will readily appreciate from the
present disclosure, processes, manufacture, composition of matter,
methods, or steps, presently existing or later to be developed that
perform substantially the same function or achieve substantially
the same result as the corresponding embodiments described herein
may be utilized according to the present disclosure.
[0133] It will be appreciated by those skilled in the art that
various modifications and alterations of the invention can be made
without departing from the broad scope of the appended claims. Some
of these have been discussed above and others will be apparent to
those skilled in the art.
* * * * *