U.S. patent application number 13/786887 was filed with the patent office on 2014-03-27 for composition for the attachment of implants to collagen or other components of biological tissue.
This patent application is currently assigned to NEW YORK SOCIETY FOR THE RUPTURED AND CRIPPLED MAINTAINING THE HOSPITAL FOR SPECIAL SURGERY. The applicant listed for this patent is New York Society for the Ruptured and Crippled Maintaining the Hospital for Special Surgery, The Texas A&M University System, The Trustees of Princeton University. Invention is credited to Axel Magnus Hook, Casey Marie Jones, Suzanne A. Maher, Brooke Hageman Russell, Jeffrey Schwartz.
Application Number | 20140088007 13/786887 |
Document ID | / |
Family ID | 46516080 |
Filed Date | 2014-03-27 |
United States Patent
Application |
20140088007 |
Kind Code |
A1 |
Maher; Suzanne A. ; et
al. |
March 27, 2014 |
COMPOSITION FOR THE ATTACHMENT OF IMPLANTS TO COLLAGEN OR OTHER
COMPONENTS OF BIOLOGICAL TISSUE
Abstract
The present invention relates to a novel composition comprising
an implant, scaffold or construct bound to a biological or chemical
moiety. The bound moiety has the ability to bind to a component of
the extracellular matrix of biological tissue, allowing the implant
to be bound to the biological tissue in a short period of time
after implantation. The invention also relates to the use and
manufacture of this novel composition, as well as a novel use for
the protein CNA.
Inventors: |
Maher; Suzanne A.; (Highland
Lakes, NJ) ; Schwartz; Jeffrey; (Princeton, NJ)
; Hook; Axel Magnus; (Houston, TX) ; Russell;
Brooke Hageman; (Pearland, TX) ; Jones; Casey
Marie; (Portland, OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hospital for Special Surgery; New York Society for the Ruptured and
Crippled Maintaining the
The Trustees of Princeton University;
The Texas A&M University System; |
|
|
US
US
US |
|
|
Assignee: |
NEW YORK SOCIETY FOR THE RUPTURED
AND CRIPPLED MAINTAINING THE HOSPITAL FOR SPECIAL SURGERY
New York
NY
THE TEXAS A&M UNIVERSITY SYSTEM
College Station
TX
THE TRUSTEES OF PRINCETON UNIVERSITY
Princeton
NJ
|
Family ID: |
46516080 |
Appl. No.: |
13/786887 |
Filed: |
March 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13353952 |
Jan 19, 2012 |
8440618 |
|
|
13786887 |
|
|
|
|
61434144 |
Jan 19, 2011 |
|
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|
Current U.S.
Class: |
514/7.6 ;
514/1.1; 514/13.6; 514/17.2 |
Current CPC
Class: |
A61K 47/64 20170801;
A61P 19/00 20180101; A61P 41/00 20180101; A61P 21/00 20180101; A61P
7/04 20180101; A61K 47/6957 20170801; A61K 38/39 20130101; A61K
38/18 20130101; A61K 38/17 20130101; A61K 38/36 20130101 |
Class at
Publication: |
514/7.6 ;
514/1.1; 514/17.2; 514/13.6 |
International
Class: |
A61K 38/39 20060101
A61K038/39; A61K 38/36 20060101 A61K038/36; A61K 38/18 20060101
A61K038/18; A61K 38/17 20060101 A61K038/17 |
Claims
1-24. (canceled)
25. A composition comprising an implant and a biological moiety
derived from bacteria or a chemical moiety, wherein the biological
or chemical moiety is bound to the implant and has the ability to
bind to a component of the extracellular matrix of a biological
tissue, whereby the implant and the biological tissue become
attached upon introduction of the composition to biological
tissue.
26. The composition of claim 25, wherein the biological moiety is a
protein, protein sub-domain or mutated protein.
27. The composition of claim 26 wherein the protein, protein
sub-domain or mutated protein has an increased ability to bind to
the component of the extracellular matrix of the biological
tissue.
28. The composition of claim 26, wherein the protein is a mammalian
collagen binding protein.
29. The composition of claim 26, wherein the protein is derived
from Staphylococcus aureus, Enterococcus faecalis, or Streptococcus
mutans.
30. The composition of claim 25, wherein the biological tissue is
musculoskeletal tissue.
31. The composition of claim 25, wherein the implant is comprised
of a non-biodegradable, partly degradable or fully degradable
polymer.
32. The composition of claim 25, wherein the implant is comprised
of poly(vinyl) alcohol.
33. The composition of claim 25, wherein the implant is porous.
34. The composition of claim 25, wherein the implant allows for the
migration of cells into the implant.
35. The composition of claim 25, wherein the implant is fibrin or
fibrinogen.
36. The composition of claim 25, further comprising a growth factor
or chemoattractant.
37. A composition comprising an implant and a biological moiety
derived from bacteria or a chemical moiety, wherein the biological
or chemical moiety is bound to the implant and has the ability to
bind to collagen in biological tissue, whereby the implant and the
biological tissue become attached upon introduction of the
composition to biological tissue.
38. A method of using the composition of claim 37 for the
treatment, repair or replacement of musculoskeletal tissue, by
implantation of the composition into a subject in need thereof.
39. The method of claim 37, wherein the subject is a mammal.
40. The method of claim 39, wherein the mammal is a human.
41. A method of using the composition of claim 37 for the
treatment, repair or replacement of musculoskeletal tissue, by
implantation of the composition into a subject in need thereof.
42. A method for the manufacture of the composition comprising an
implant and a biological or chemical moiety of claim 25, comprising
the steps of: a. lyophilizing the implant for a period of time
sufficient to remove all of the water from the implant without
collapsing the porous structure; b. exposing the implant to a vapor
of zirconium tetra(tert-butoxide); c. heating the implant; d.
agitating the implant in the presence of zero grade nitrogen; e.
re-exposing the implant to a vapor of zirconium
tetra(tert-butoxide); f. heating and agitating the implant in the
presence of zero grade nitrogen; g. exposing the implant to
11-phosphonoundecanol; h. submerging the implant in p-nitrophenyl
chloroformate dissolved in tetrahydrofuran; i. adding
diisopropylethylamine to the solution containing the implant; and
j. removing the implant; wherein the resulting composition
comprises the implant and a chemical moiety bound to the
implant.
43. The method of claim 42, further comprising the step of soaking
the implant in a solution of a biological or chemical moiety for a
period of time sufficient for the chemical or biological moiety to
bind to the implant.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation application of
U.S. patent application Ser. No. 13/353,952, filed Jan. 19, 2012,
entitled "A Composition for the Attachment of Implants to Collagen
or Other Components of Biological Tissue", now issued as U.S. Pat.
No. 8,440,618, issued May 14, 2013, which claims priority to U.S.
patent application Ser. No. 61/434,144, filed Jan. 19, 2011, which
is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention is in the field of implants, more
specifically a composition in which an implant will attach to a
component of the extracellular matrix in a biological tissue, in a
short period of time after implantation into a subject. The present
invention is also a method for use and manufacture of the
composition as well as a novel use for the protein, CNA, derived
from the collagen adhesion gene of Staphylcoccus aureus.
BACKGROUND OF THE INVENTION
[0003] Articular cartilage is a hydrated and lubricated joint
tissue that allows for the relative movement of opposing joint
surfaces under high loads (Buckwalter et al. (1998)). A sparse
distribution of chondrocytes reside in the dense extracellular
matrix of the tissue, with components such as collagen,
proteoglycan and water inhomogenously dispersed through the depth
of the tissue.
[0004] Mature articular cartilage does not possess an intrinsic
ability to heal, since it is avascular and lacks a source of
mesenchymal cells (Buckwalter et al. (1998)). Therefore, small
focal cartilage defects can propagate unchecked to include the
entire joint, eventually leading to osteoarthritis (OA) (Buckwalter
et al. (1998)), a condition which affects as many as 27 million
Americans with societal costs greater than $15 billion annually
(Woolf et al. (2003): Gelber et al. (2000)). Current operative
procedures for the treatment of articular cartilage damage
generally fall into four categories: 1. nontransplant salvage
operations such as abrasion arthroplasty; 2. mosaicplasty in which
a cartilage-bone plug is transplanted into a joint which is
minimally weight bearing; 3. reimplantation of autogenously
isolated and expanded cells; and 4. the implantation of allografts
(Maher et al. (2007); Szerb et al. (2005); Freedman et al. (2003);
Brittburg et al. (2003); Gross (2003)). However, all of these
techniques have their limitations, and do not prevent the
progression of the osteoarthritis, which often propagates from the
focal defect. Furthermore, over 33% of those affected by arthritis
are under age 65. Accordingly, the number of young patients with
total knee replacement, the end stage treatment for arthritis, is
increasing. Performance of joint replacements in younger patient
populations is less satisfactory than for older patients,
oftentimes leading to multiple revision surgeries, each with
successively diminishing longevity.
[0005] The problem with many of the surgical approaches and
implantable materials that have been developed thus far, is the
inability to integrate with the native tissue. For example,
microfracture, the most commonly used clinical procedure for the
treatment of full-thickness defects, results in poor integration
between the fibrocartilage tissue that fills the defect site and
the surrounding host tissue hyaline cartilage (Lane et al. (2010);
Fortier et al. (2010); Gill et al. (2005); Hoemann et al. (2005);
Watanabe et al. (2009); Hattori et al. (2008); LaPrade et al.
(2008); Morisset et al. (2007); Kreuz et al. (2006)). Osteochondral
autograft transfer is another technique developed to fill
osteoarticular defects in weight bearing regions of the knee (Bobic
(1996); Hangody et al. (1998)). However, chondrocyte death at the
margins of the autograft (Evans et al. (2004); Zhang et al. (2005);
Hunziker et al. (2003); Enders et al. (2010)) and persistent gaps
between the graft and the host tissue (Kock et al. (2004); Lane et
al. (2004); Williams et al. (2007); Marquass et al. (2010)) have
been shown to lead to poor graft durability over time (Solheim et
al. (2010)). In cases where implants are used to fill the defect,
margin integration is frequently characterized by gaps and
fissuring in histologic sections (Schafer et al. (2002); Niederauer
et al. (2000); Wegener et al. (2010); Nehrer et al. (1998); Jiang
et al. (2007); Ito et al. (2005); Wang et al. (2010)).
[0006] Efforts to create a mechanically stable interface between an
implant and the host articular cartilage have explored the use of
partial enzymatic digestion of the host tissue (Obradovic et al.
(2001); Hunziker et al. (1998)) and the release of chemotactic
agents to increase the number of matrix generating cells at the
interface (Pabbruwe et al. (2009); Fortier et al. (2002)). Such
approaches may help to reinforce the boundary between the scaffold
and the host tissue as a function of time, but they do not address
the problems associated with an initially unstable interface. Newer
approaches rely on the use of an adhesive agent as an intermediary
that chemically binds the native tissue to the implant in an
attempt to immediately "glue" the scaffold to the surrounding
native cartilage. One such example involves using a functionalized
chondroitin sulphate paste to create a covalent bond between a
biomaterial and proteins in articular cartilage (Wang et al.
(2007); Strehin et al. (2010)). While the technology has been
demonstrated to increase the interfacial strength and percent
tissue fill in scaffold implanted cartilage defects, the addition
of yet another interface (that between the glue and the implant and
the implant and the cartilage) is far from ideal.
[0007] Therefore, there is a need in the art for a more reliable
method to treat patients with focal defects, especially young
active ones, early in the course of the problem, thus delaying or
eliminating the need for total joint replacement, and a real need
in the art for composition and method which increases focal
strength and integration of an implant in a short period of time
after implantation.
SUMMARY OF THE INVENTION
[0008] The present invention overcomes the problems in the art by
providing for a novel composition comprising an implant, construct
or scaffold to which a biological or chemical moiety is bound, such
biological or chemical moiety having the ability to bind to a
component of the extracellular matrix of the host tissue upon
implantation. Upon implantation, the moiety of the composition
would allow the implant to integrate with the extra-cellular matrix
components of the host tissue in a short period of time.
[0009] In a preferred embodiment, the moiety would bond with
collagen, thus, any tissue that contains collagen in its
extracellular matrix is a candidate for implantation of the
composition. In a preferred embodiment, the composition is suitable
for implantation into a mammal, to treat, repair or replace defects
and/or injury to musculoskeletal tissue including bone, tendon,
ligaments, cartilage and the discs of the spine. In a preferred
embodiment the implant is used to stabilize a chrondral defect,
which is a defect in the articular cartilage at the end of the
bones. In a most preferred embodiment, the chondral defect is found
in the knee.
[0010] In a preferred embodiment, the moiety is chemical, and in a
most preferred embodiment, contains a chemically reactive group,
such as a carbonate ("open carbonate" or "OC").
[0011] In another preferred embodiment, the moiety is biological.
Biological moieties would be derived from living organisms or
through protein engineering, and could include, but are not limited
to, proteins, protein sub-domains, and mutated proteins with
altered affinity for a ligand, in particular, collagen. One source
for biological moieties would be bacteria, including but not
limited to Staphylococcus aureus, Enterococcus faecalis, and
Streptococcus mutans. Other sources would be mammalian collagen
binding proteins, such as decorin. A preferred biological moiety is
a protein derived from Staphylococcus aureus, encoded by the
collagen adhesion gene, CNA.
[0012] The implant, scaffold, or construct of the novel composition
can be made of non-degradable, partially degradable, or fully
degradable polymer. A preferred material for the implant is
poly(vinyl) alcohol ("PVA"). It is also preferred that the implant
be porous, and allow for and facilitate the migration of cells into
the implant. The implant can also be fibrinogen or fibrin.
[0013] Another embodiment of the present invention is a method to
manufacture the composition by funetionalizing the implant with the
moiety.
[0014] Another embodiment of the present invention is a method for
the treatment, repair or replacement of biological tissue, in a
subject in need thereof, by implantation of the composition. In a
preferred embodiment, the biological tissue is musculoskeletal
tissue.
[0015] Yet another embodiment of the present invention is a novel
use for the CNA protein derived from Staphylococcus aureus. This
novel use is based upon the ability of CNA to bind to collagen in
tissue which contains collagen. This ability allows CNA to be used
as a binder or connector between host tissue found in a subject and
a foreign object or implant being used to treat or diagnose the
subject.
[0016] Another embodiment of the present invention is a novel use
for reactive organic carbonate (open carbonate) groups. Again, this
novel use is based upon the ability of the reactive organic
carbonate groups to bind to collagen or other amino group side
chain-containing proteins or molecules in tissue. This ability
allows the reactive organic carbonate groups to be used as a binder
or connector between host tissue found in a subject and a foreign
object or implant being used to treat or diagnose the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] For the purpose of illustrating the invention, there are
depicted in drawings certain embodiments of the invention. However,
the invention is not limited to the precise arrangements and
instrumentalities of the embodiments depicted in the drawings.
[0018] FIG. 1 are fluorescence microscopy images showing that CNA
binds to cartilage (FIGS. 1(A) and 1(B)) and PVA scaffolds (FIGS.
1(C) and 1(D)). FIG. 1(A) depicts Texas-Red labeled CNA attached to
full thickness bovine articular cartilage as seen by the red
fluorescence using fluorescent microscopy (red fluorescence). FIG.
1(B) depicts anti-hexa-histidine tag antibodies bound to CNA
detected with immunohistochemistry on bovine articular cartilage.
FIGS. 1(C) and (D) show Texas-Red labeled CNA covalently bonded to
the scaffold as seen by the red fluorescence using fluorescent
microscopy.
[0019] FIG. 2 is a representation of the series of steps required
to functionalize the scaffolds.
[0020] FIG. 3 shows fluorescence microscopy images of FITC-labeled
collagen II incubated with PVA scaffolds functionalized with CNA,
OC (open carbonate), BSA (bovine serum albumin), and UC
(untreated). The presence of collagen II is only detected on the
CNA and OC scaffolds, indicating that those two scaffolds have the
ability to bind collagen.
[0021] FIG. 4 shows images of PicroSirius staining of PVA
scaffolds. FIG. 4(A) shows a PVA scaffold functionalized with CNA
incubated with collagen in suspension, FIG. 4(B) shows a PVA
scaffold functionalized with CNA but not incubated with collagen,
and FIG. 4(C) shows a non-functionalized scaffold incubated with
collagen.
[0022] FIG. 5(A) shows an example of a bovine articular cartilage
discs which were biopsy punched forming a central cylindrical 3.5
mm defect. A 5 mm diameter scaffold was press-fitted into the
central defect. FIG. 5(B) shows the push-out test set-up. FIG. 5(C)
is a graph of the interface strength of each group of
functionalized scaffolds (UC, BSA, OC, and CNA), at Day 0 and Day
21.
[0023] FIG. 6(A) is a graph depicting the GAG content as measured
by DMMB assay and nounalized to the wet weight of the scaffold, for
each group of functionalized scaffolds (UC, BSA, OC, and CNA) at
Day 21. FIG. 6(B) is a graph depicting the DNA content as measured
by a pico-green assay and normalized to the wet weight of the
scaffold, for each group of functionalized scaffolds (UC, BSA, OC,
and CNA) at Day 21.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The current invention is a novel composition comprising an
implant, scaffold or construct bound to a biological or chemical
moiety, which upon implantation into a subject, the moiety binds to
a component of the extracellular matrix of the host tissue.
[0025] The current invention is also a method of manufacturing the
novel composition.
[0026] The current invention is also a method of treating,
repairing or replacing biological tissue, preferably
musculoskeletal tissues, by implanting the biocompatible
composition into a subject, in need thereof.
[0027] The current invention is also a method of using the
bacterial protein encoded by the collagen adhesion gene of
Staphylococcus aureus, known as CNA. The novel use of CNA is based
upon its ability to bind to collagen in tissue which contains
collagen. This ability allows CNA to be used as a binder or
connector between host tissue found in a subject and a foreign
object or implant being used to treat or diagnose the subject.
[0028] The current invention is also a method for using reactive
organic carbonate (open carbonate) groups. Again, this novel use is
based upon the ability of the reactive organic carbonate groups to
bind to collagen or other amino group side chain-containing
proteins or molecules in tissue. This ability allows the reactive
organic carbonate groups to be used as a binder or connector
between host tissue found in a subject and a foreign object or
implant being used to treat or diagnose the subject.
DEFINITIONS
[0029] The terms used in this specification generally have their
ordinary meanings in the art, within the context of this invention
and the specific context where each term is used. Certain terms are
discussed below, or elsewhere in the specification, to provide
additional guidance to the practitioner in describing the methods
of the invention and how to use them. Moreover, it will be
appreciated that the same thing can be said in more than one way.
Consequently, alternative language and synonyms may be used for any
one or more of the terms discussed herein, nor is any special
significance to be placed upon whether or not a term is elaborated
or discussed herein. Synonyms for certain terms are provided. A
recital of one or more synonyms does not exclude the use of the
other synonyms. The use of examples anywhere in the specification,
including examples of any terms discussed herein, is illustrative
only, and in no way limits the scope and meaning of the invention
or any exemplified term. Likewise, the invention is not limited to
its preferred embodiments.
[0030] The term "biocompatible" as used in the application means
capable of coexistence with living tissues or organisms without
causing harm.
[0031] The term "extracellular matrix" as used in the application
means the substance of a tissue outside and between cells.
[0032] The term "moiety" as used in the application means part of a
composition that exhibits a particular set of chemical and
pharmacologic characteristics. "Biological moieties" are those
which derive from living organisms or through protein
engineering.
[0033] The terms "implant", "construct", and "scaffold" are used
interchangeably throughout this application and means any material
inserted or grafted into the body that maintains support and tissue
contour.
[0034] The term "porous" as used in the application means having
pores, which are defined as a minute opening.
[0035] The term "subject" as used in this application means an
animal with an immune system such as avians and mammals. Mammals
include canines, felines, rodents, bovines, equines, porcines,
ovines, and primates. Avians include, but are not limited to,
fowls, songbirds, and raptors. Thus, the invention can be used in
veterinary medicine, e.g., to treat companion animals, farm
animals, laboratory animals in zoological parks, and animals in the
wild. The invention is particularly desirable for human medical
applications.
[0036] The term "in need thereof" would be a subject known or
suspected of having an injury to or defect in any tissue,
preferably musculoskeletal tissue, including but not limited to,
cartilage, bone, tendon, ligaments, and the discs of the spine, and
other tissues that contain collagen.
[0037] The terms "treat", "treatment", and the like refer to a
means to slow down, relieve, ameliorate or alleviate at least one
of the symptoms of the defect or injury or reverse the defect or
injury after its onset.
[0038] A "chondral defect" is defined as a defect in the articular
cartilage at the end of the bones.
[0039] The term "polymer" means a large molecule composed of
repeating structural units often connected by covalent chemical
bonds. Polymers can be natural or synthetic.
[0040] The terms "mutant" and "mutation" mean any detectable change
in genetic material, e.g. DNA, or any process, mechanism, or result
of such a change. This includes gene mutations, in which the
structure (e.g. DNA sequence) of a gene is altered, any gene or DNA
arising from any mutation process, and any expression product (e.g.
protein or enzyme) expressed by a modified gene or DNA
sequence.
Biological or Chemical Moiety
[0041] Microbial surface component recognizing adhesive matrix
molecules or MSCRAMMs allow bacteria or other entities to attach to
an extracellular matrix component in a tissue. Staphylococcus
aureus is an aggressive bacterium that infects many tissues
including cartilage. Its virulence is mediated by its ability to
attach very strongly to collagen. The bacterium utilizes the
MSCRAMM which is encoded by the CNA gene, known as the CollageN
Adhesion protein or CNA (Mohamed et al. (1999); Switalski et al.
(1989); Patti et al. (1992); Xu et al. (2004); Patti et al. (1994);
Zong et al. (2005)).
[0042] CNA is a well characterized protein that has affinity for
collagen type I, collagen type II (a major constituent of the
extracellular matrix of articular cartilage), and collagen-like
peptides (Switalski et al. (1989); Patti et al. (1992); Xu et al.
(2004); Zong et al. (2005)). The full length CNA is approximately
135 kDal and contains an N-terminal signal sequence, an A region
containing N1, N2, and N3 domains, B repeats, and a cell wall
anchoring region. The CNA (CNA35) minimal binding region
demonstrates the highest affinity for collagen (Xu et al. (2004);
Zong et al. (2005)). CNA35 constitutes the N1 and N2 domains
separated by a linker sequence with each domain exhibiting an
IgG-like fold. Biochemical assays in combination with x-ray
crystallography suggest a mechanism of binding where the N2 domain
initiates contact with the collagen triple helix; subsequently the
linker and N1 domain wrap around the helix using a so-called "hug
mechanism" (Zong et al. (2005)).
[0043] Because CNA is well-characterized and has been successfully
cloned, expressed using a vector in an E. coli host, and purified,
there is an ability to mass produce purified recombinant CNA.
[0044] The current invention is based upon the strong binding
affinity of CNA to collagen. One embodiment of the current
invention is the novel use of the protein encoded by the CNA gene
of Staphylococcus aureus. The CNA can be used as a binder or
connector of host tissue found in a subject that contains collagen
and a foreign object or implant being used to treat or diagnose the
subject. While recombinant CNA has been extensively tested with
collagen monomer fibers in suspension, its ability to bind to
collagen in intact pieces of cartilage had not been tested to date.
As shown in Example 2, recombinant CNA can bind to collagen in
intact pieces of cartilage.
[0045] Another embodiment of the present invention involves
attaching one end of CNA to the periphery of an implant, such that
upon implantation, the other end can attach to collagen in
articular cartilage, the implant would immediately attach to the
collagen, preferably collagen II, in articular cartilage or any
other tissue containing collagen.
[0046] As shown in Examples 4 and 5, implant-bound CNA can bind to
collagen type II. Moreover, the implant-bound CNA increased the
cartilage-implant interface strength at Day 0 as compared to
implants functionalized with another protein, bovine serum albumin,
and those unfunctionalized, without adversely affecting the ability
of the cells to migrate into the scaffold (Example 6). There was no
significant difference in the number of cells that migrated into
the scaffold, in the amount of new matrix deposited, or in the
histological appearance of the samples as a function of the
treatment, suggesting that the presence of CNA does not have an
adverse effect on chondrocyte migration. Because the CNA
significantly increases the Day 0 interfacital strength between the
implant and articular cartilage, without adversely affecting the
cellular response, the CNA can be used to create a stable interface
across which cells can migrate and lay down a matrix.
[0047] While CNA is the exemplified moiety, any chemical or
biological moiety that has the ability to attach to collagen can be
used. Examples of chemical moieties that could be used to bind
collagen include, but are not limited to, organic carbonate,
--OC(.dbd.O)O--. As shown in Example 4, implants functionalized
with "open carbonate groups", where the reactive site remained free
to interact with any amino group side chain-containing protein or
molecule, also resulted in an ability to bind collagen type II.
This also resulted in an increased interfacial strength at Days 0
and 21, with no change in the number of cells that migrated into
the scaffold, in the amount of new matrix deposited, or in the
histological appearance.
[0048] Biological moieties would be derived from living organisms
or through protein engineering, and could include, but are not
limited to, proteins, protein sub-domains, and mutated proteins
with altered affinity for a ligand, in particular, collagen. One
source for biological moieties would be bacteria, including but not
limited to Staphylococcus aureus, Enterococcus faecalis, and
Streptococcus mutans. Other sources would be mammalian collagen
binding proteins, such as decorin. In one embodiment, proteins such
as a MSCRAMM or CNA, can be attached to the polymer or implant
directly.
[0049] Moreover, as would be understood by one of skill in the art,
chemical and biological moieties can be used which have the ability
to bind to another component of the extracellular matrix of the
host tissue, other than collagen. Such components include, but are
not limited to, fibronectin and laminin. Moieties that would have
the ability to bind to these components, include, but are not
limited to, FnBPAIB, BBK32, and Lmb.
[0050] It will also be understood by a person of skill in the art
that the moieties can be modified to have an increased binding with
collagen or another component of the extracellular matrix of the
host tissue. With regard to biological moieties, especially
recombinant proteins such as CNA, it is within the skill of those
in the art to modify the protein via recombinant DNA or genetic
engineering techniques in order to obtain a protein with increased
binding activity.
Implants
[0051] Laboratory investigations and preclinical animal studies
have most recently focused on the use of biodegradable matrix
scaffolds, alone and in combination with chondrogenic cells, in
order to improve the quality of cartilage repair tissue after
surgery (Kuo et al. (2006)). Both natural and synthetic polymers
have been fabricated for use as cell-seeded scaffolds, the
chemistry and biology of which has taken a variety of forms,
including fibrous structures, porous sponges, woven or non-woven
meshes, and hydrogels (Kuo et al. (2006); Williams and Gamradt
(2008); Vacanti et al. (1991); Cima et al. (1991)). Integration
with the surrounding native cartilage remains a significant
challenge for all implants. Thus, any implant, including but not
limited to the ones referenced above, suitable for implantation
into tissue, preferably musculoskeletal tissue, can be used in the
composition. The implant can be made of non-degradable, partly
degradable, or fully degradable natural or synthetic polymer
depending upon the use and location of the implant. The implant can
also contain other factors immobilized on the surface, such as
those that support cell migration and/or matrix deposition, such as
growth factors and chemoattractants. One of skill in the art can
determine which type of polymer is best suited for the desired use
and location of the implant.
[0052] One preferred implant is fibrinogen or fibrin.
[0053] Another preferred implant is made of poly(vinyl alcohol) or
PVA. A preferred implant is also porous, and more preferably would
allow the inward migration of cells. A porous PVA scaffold for the
purposes of replacing, repairing or treating defects or injury in
musculoskeletal tissue, preferably cartilage, has been described in
U.S. patent application Ser. No. 13/349,365, incorporated herein by
reference in its entirety. It has been shown that cells can migrate
into and remain viable within the construct.
[0054] The implant or scaffold can be chemically modified so that
the biological or chemical moiety capable of binding to the
extracellular matrix of the host cell can become attached to the
implant, providing immediate attachment between the implant and the
host tissue. In a preferred embodiment, the protein, CNA, can be
attached to a PVA implant. When implanted, the CNA will adhere to
the exposed fibrillar collagen type II in the cartilage and provide
immediate attachment between the implant and articular cartilage.
The porous PVA implant will readily accept the inward migration of
cells to further enhance integration, but the initial adhesion
between the implant and cartilage is not contingent upon the cell
migration. Another preferred embodiment, carbonate groups are
attached to a PVA implant, and supply the same immediate attachment
between the implant and articular cartilage.
[0055] CNA was attached to the previously described PVA scaffold
using a novel method to insure that the protein would be anchored
using the N-terminus. This type of anchoring will not interfere
with the CNA ability to bind to collagen. The PVA implant is
chemically modified or functionalized to have the CNA protein or
open carbonate attached by the sequence of steps set forth in FIG.
2 and described in detail in Example 2. One of skill in the art can
easily modify the method in order to obtain an implant with any
biological or chemical moiety that binds to a host tissue. In
particular, the R group shown in FIG. 2, (5) and (6), can be
changed to attach the desired chemical or biological moiety using
standard methods of organic synthesis. The adhesion layer used to
bond the OC can be applied to any organic or inorganic polymer that
contains O, N, or S ligating groups.
Use of the Composition
[0056] The composition comprising an implant, construct or scaffold
to which a chemical or biological moiety is bound of the current
invention can be used to treat, replace or repair defects and/or
injuries in various tissues, in a subject in need thereof,
preferably a mammal, and most preferably a human. In a preferred
embodiment, the tissue would contain collagen in the extracellular
matrix and most preferably is musculoskeletal tissue.
Musculoskeletal tissue contemplated to be treated, replaced or
repaired includes bone, tendon, ligaments, cartilage and the discs
of the spine. In a preferred embodiment, the biocompatible
composition is used to replace focal defects in articular
cartilage. In a most preferred embodiment, the defect is found in
the knee. The biocompatible composition, due to the chemical or
biological moiety, attaches immediately to the host tissue, and
provides increased interfacial strength to the implant immediately
upon implantation. In a preferred embodiment, the implant allows
for the migration of cells into the implantation site, allowing for
continued increase in strength.
[0057] Those of skill in the art would appreciate that the
implants, constructs or scaffolds of the present invention may be
implanted into a subject using operative techniques and procedures,
utilizing such techniques as magnetic resonance imaging and
computer guided technology. One of skill in the art would also
appreciate that the tissue of the subject can be pre-treated prior
to implantation with any agent that would expose the collagen or
other component of the extracellular matrix to which the biological
or chemical moiety is desired to bind.
EXAMPLES
[0058] The present invention may be better understood by reference
to the following non-limiting examples, which are presented in
order to more fully illustrate the preferred embodiments of the
invention. They should in no way be construed to limit the broad
scope of the invention.
Example 1
General Materials and Methods
[0059] Expression and Purification of Recombinant CNA
[0060] The recombinant protein CNA35 was cloned into pQE30
expression plasmid and transformed into E. coli as previously
described (Zong et al. (2005)). Expression and purification of
recombinant CNA was performed as previously described (Wann et al.
(2000)). Briefly, bacterial lysates expressing an
N-terminal-hexa-histadine tagged CNA were first purified by nickel
chelate affinity chromatography using a 5-ml Hi-Trap chelating
column in a fast protein liquid chromatography system (Amersham
BioSciences, GE). Fractions, collected and analyzed by SDS-PAGE,
containing protein of expected size, were pooled and dialyzed in 25
mM Tris-HCl, pH 8 and further purified in a 5 ml Hi-Trap
Q-Sepharose column (Amersham Biosciences, GE) using a linear
gradient of 0 to 1M NaCl.
[0061] Porous Polyvinyl Alcohol (PVA) Scaffolds
[0062] Porous polyvinyl alcohol (PVA) scaffolds were manufactured
using the method described in U.S. patent application Ser. No.
13/349,365, which is incorporated herein by reference in its
entirety. In brief, rectangular collagen sponges were impregnated
with 10% weight PVA in solution with deionized water and the
construct was subjected to six freeze-thaw cycles over a period of
five days. The impregnated sponges were cored into cylindrical
geometries using a 5 mm diameter biopsy punch, and sliced to the
desired height to form cylinders. The cylindrical specimens were
digested with a collagenase solution to remove the collagen sponge
and result in an interconnected porous PVA scaffold.
[0063] Statistical Analyses
[0064] Statistical analyses were performed using Graphpad Prism
software. To test for significance, a 2-way ANOVA analysis was
conducted with a post-hoc Bonferroni post-test. The two independent
variables used were time (0 vs 21 days) and treatment type (BSA,
CNA, UC, OC).
Example 2
Recombinant CNA Can Bind Collagen Found in Intact Cartilage
Tissue
[0065] Materials and Methods
[0066] Recombinant CNA as described in Example 1 was labeled with a
fluorescent Texas-Red label as follows: CNA protein (2 mg/ml) was
first dialyzed in NaHCO.sub.3 to 100 mM final concentration (pH
8.2). The CNA protein (5 ml) was incubated with Texas-Red dye
(dissolved in DMF to 5 .mu.g/.mu.l) for 1 hour at room temperature
in a shaker covered in aluminum foil. After the labeling reaction,
the protein was dialyzed four times in 2 L phosphate buffered
saline (PBS) buffer. Thin strips of articular cartilage
approximately 1-2 mm thick (n=3) were isolated from juvenile bovine
knees, incubated with Texas-Red tagged CNA, suspended in water at a
concentration of 1 mg/mL for one hour with agitation, and washed
vigorously. Three samples were examined with fluorescence
microscopy.
[0067] Three additional articular cartilage samples were incubated
with CNA (without a Texas-Red tag), suspended in water at a
concentration of 1 mg/mL for one hour with agitation, washed,
fixed, paraffin embedded, and immunohistochemistry was performed
using an antibody to the poly-histidine tag on the CNA (Alpha
Diagnostic, TX).
[0068] Results
[0069] After one hour of incubation in the presence of Texas-Red
labeled CNA, the protein was detected on the edges of articular
cartilage using fluorescent microscopy (FIG. 1(A)). The non-tagged
CNA was also detected on the surface of the articular cartilage
using immunohistochemistry with anti-hexa-histidine tag antibodies
(FIG. 1(B)).
Example 3
PVA Scaffolds can be Functionalized with CNA
[0070] Materials and Methods
[0071] Interconnected polyvinyl alcohol (PVA) implants described in
Example 1, were functionalized using a sequential process resulting
in reactive carbonate groups on the scaffold's surface, which can
covalently bond with any amino group (Dennes et al. (2009)). The
procedure is summarized in FIG. 2 and the various processing steps
are labeled from 1 to 6.
[0072] PVA implants were lyophilized for 24 hours to remove all
water without collapsing the porous structure. They were then
exposed to vapor of zirconium tetra(tert-butoxide) (FIG. 2, (1)) as
previously described to create the interface (Dennes et al. (2009);
U.S. Application Publ. No. 2009/0104474). The reaction chamber was
then heated to 45.degree. C. and was held at this temperature for
10 minutes. The chamber was back-filled with zero grade nitrogen,
and the implant samples were then agitated to expose any surface
that had not yet been coated. The newly exposed surfaces were then
treated further with vapor of zirconium tetra(tert-butoxide) to
create the interface between the implant and the phosphonic acid
(Dennes et al. (2009); U.S. Application Publ. No.
2009/0104474).
[0073] The chamber was again heated to 45.degree. C., and was held
at this temperature for 10 minutes (FIG. 2, (2)). The chamber was
again back-filled with zero grade N.sub.2, and the implants were
quickly transferred into a 0.1 mM solution of 11-phosphonoundecanol
(FIG. 2, (3)) in ethanol. After 12 hours the implants were removed
from this solution, sonicated in ethanol, and dried in vacuo to
give the surface-bound hydroxyalkylphosphonate. p-Nitrophenyl
chloroformate (80 mg, 0.4 mmol) was dissolved under argon in 60 mL
dry tetrahydrofuran in a 100 mL three-necked round bottom flask
equipped with a dry stir bar and the activated implants (FIG. 2,
(4)) were then submerged in the solution. Dry diisopropylethylamine
(0.75 mL, 4 mmol) was added, and the suspension was stirred for 45
minutes. The implants were removed from this suspension and washed
briefly with ethanol. These steps resulted in an implant
functionalized with a group (called "open carbonates" or "OC") that
can react with any nucleophile in solution.
[0074] The implants were then soaked in a solution of CNA in water
(pH 8.5) for 24 hours (FIG. 2, (5)), or BSA in water (FIG. 2, (6)),
or not soaked at all. All samples were removed from the solution
and washed sequentially with water and ethanol.
[0075] These steps resulted in the following groups: [0076] (i) an
organophosphonate terminated with CNA (CNA); [0077] (ii) the same
phosphonate terminated with Bovine Serum Albumin (BSA), used as a
control for functionalizing with a protein; [0078] (iii) the
organophosphonate terminated with organic carbonate groups that are
free to react with any nucleophile in solution (called "open
carbonates," OC); and, [0079] (iv) untreated control (UC)
[0080] Three samples functionalized with Texas-Red labeled CNA were
frozen sectioned and imaged using fluorescent microscopy.
[0081] Results
[0082] As shown in FIGS. 1(C) and 1(D), all the PVA scaffolds that
were functionalized with Texas-Red labeled CNA, were found to have
florescent edges indicating that the process successfully bound CNA
to the walls of the scaffold.
Example 4
The Functionalized PVA Scaffolds can Bind Collagen
[0083] Materials and Methods
[0084] The PVA scaffolds (five from each of groups (i), (ii),
(iii), and (iv), as described in Example 3), were incubated for
three hours with a solution of collagen type II isolated from
bovine articular cartilage and labeled with FITC (Sigma, Mo.) at
0.25 mg/mL. Each scaffold was washed with three centrifugation
steps, frozen sectioned and imaged using fluorescence microscopy.
Scaffolds that were not incubated with the collagen were also
imaged to ensure that there was no auto-fluorescence. Images were
captured using a microscope with exposure time consistent between
groups by a blinded observer.
[0085] Results
[0086] The CNA-functionalized group of scaffolds bound collagen
type II as demonstrated by a qualitative detection of the FITC tag
on the collagen via fluorescence microscopy (FIG. 3). This result
indicates that the processing steps to which the scaffold and CNA
were subjected (Example 3) did not interfere with the ability of
CNA to bind collagen. Surprisingly, the OC group also demonstrated
an ability to bind collagen type II. The UC and BSA groups did not
bind collagen (FIG. 3).
Example 5
Further Proof that the Funtionalized PVA Scaffolds can Bind to
Collagen
[0087] Material and Methods
[0088] The PVA scaffolds functionalized in Example 3 were incubated
in a solution of collagen type II isolated from bovine articular
cartilage in phosphate buffered saline (PBS) for 4 hours. The
scaffolds were removed from solution, vigorously washed in PBS, and
stained with PicroSirius Red (Polysciences Inc., Warrington, Pa.),
which stains collagen red. The samples were then fixed, paraffin
embedded, sliced, and examined under a microscope.
[0089] Results
[0090] As shown in FIG. 4, the CNA functionalized PVA implant
incubated with collagen is stained red by the PicroSirius Red (FIG.
4(A)). The CNA functionalized implant not incubated with collagen
(FIG. 4(B)) and a non-functionalized PVA implant incubated with
collagen (FIG. 4(C)) are not stained by the PicroSirius Red,
showing that only the PVA implant functionalized with CNA bound to
the collagen.
Example 6
Binding Strength of Functionalized PVA Scaffolds and Cartilage at
Day 0
[0091] Materials and Methods
[0092] Middle zone cartilage discs were isolated from juvenile
bovine knees (10 mm diameter, 2 mm thick) and perforated in the
center with a 3.5 mm diameter biopsy punch. Porous PVA scaffolds
with a 5 mm diameter functionalized with CNA, BSA, OC, or US, as
described in Example 3 (n=20 per group) were press fit into the
central hole of the cartilage explants as shown in FIG. 5(A). Each
bovine cartilage disc with the implant was incubated for four (4)
hours at 37.degree. C. in culture media and then subject to a
push-out test.
[0093] To perform the push-out test, each sample was mounted on the
base of a custom-built testing machine (as shown in FIG. 5(B)) and
a stainless steel indenter used to push on the implant at a rate of
0.01 mm/s until the implant was completely pushed out of the
cartilage ring (Bravenboer et al. (2004)). The maximum load was
recorded and normalized to the surface area of the interface for
each sample to compute maximum stress.
[0094] Results
[0095] As shown in FIG. 5(C), the interface strength on Day 0 was
approximately three times higher for the implants from the CNA and
OC groups than the BSA and UC groups. These differences were
statistically significant. There is no significant difference
between the push-out strength of the CNA and OC groups or between
the BSA and UC groups.
Example 7
Interfacial Strength of PVA Scaffolds and Cartilage and
Biocompatibility at Day 21
[0096] Materials and Methods
[0097] An additional 13 scaffold-cartilage constructs per group
were cultured in 30 mL ADMEM/F12 with 100 nM dexamethasone, 50
.mu.g/mL ascorbate-2-phosphate, and antibiotics (Sigma Aldrich) for
21 days, after which they were subjected to a push-out test where
the maximum load was recorded. The scaffolds were digested using
proteinase K (Sigma, Mo.), and assessed for glycosaminoglycan (GAG)
content using a dimethylmethylene blue assay (DMMB) and DNA content
using a quant-iT kit Pico green assay (Invitrogen, CA).
[0098] Results
[0099] As also shown in FIG. 5(C), on Day 21, the interface
strength of the BSA was significantly higher than that of the CNA
group. There was also a significant decrease in the push-out
strength of the interface of the CNA group compared to the OC
group. The BSA group had significantly higher push-out strength on
Day 21 versus that of the CNA group. Also on Day 21, there was a
significant decrease in the push out strength of the interface of
the CNA group compared to that of the OC group.
[0100] There were no significant differences in GAG (FIG. 6(A))
content, or DNA content (FIG. 6(B)) after 21 days of incubation
between the groups.
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