U.S. patent application number 14/960206 was filed with the patent office on 2016-08-04 for hyaluronic acid-binding synthetic peptidoglycans, preparation, and methods of use.
This patent application is currently assigned to Symic Biomedical, Inc.. The applicant listed for this patent is Symic IP, LLC. Invention is credited to Jonathan C. Bernhard, John Eric Paderi, Alyssa Panitch, Shaili Sharma.
Application Number | 20160222064 14/960206 |
Document ID | / |
Family ID | 47218093 |
Filed Date | 2016-08-04 |
United States Patent
Application |
20160222064 |
Kind Code |
A1 |
Panitch; Alyssa ; et
al. |
August 4, 2016 |
HYALURONIC ACID-BINDING SYNTHETIC PEPTIDOGLYCANS, PREPARATION, AND
METHODS OF USE
Abstract
This invention pertains to the field of hyaluronic acid-binding
synthetic peptidoglycans and methods of forming and using the
same.
Inventors: |
Panitch; Alyssa; (W.
Lafayette, IN) ; Bernhard; Jonathan C.; (South Bend,
IN) ; Paderi; John Eric; (San Francisco, CA) ;
Sharma; Shaili; (Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Symic IP, LLC |
San Francisco |
CA |
US |
|
|
Assignee: |
Symic Biomedical, Inc.
San Francisco
CA
|
Family ID: |
47218093 |
Appl. No.: |
14/960206 |
Filed: |
December 4, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14119341 |
Nov 21, 2013 |
9217016 |
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PCT/US12/39404 |
May 24, 2012 |
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14960206 |
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61550621 |
Oct 24, 2011 |
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61489602 |
May 24, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/78 20130101;
A61P 19/08 20180101; A61K 38/39 20130101; A61L 27/3654 20130101;
A61P 29/00 20180101; A61P 19/02 20180101; C07K 9/00 20130101; C07K
14/4725 20130101; A61K 38/00 20130101; A61L 27/54 20130101; A61K
38/1709 20130101; C08B 37/0072 20130101; C08B 37/0069 20130101;
A61L 27/48 20130101 |
International
Class: |
C07K 9/00 20060101
C07K009/00; C08B 37/00 20060101 C08B037/00 |
Claims
1. A synthetic peptidoglycan comprising a glycan and 1 to 20
synthetic peptides conjugated to the glycan, wherein each synthetic
peptide is 5 to 40 amino acids in length and comprises a hyaluronic
acid-binding amino acid sequence and wherein the synthetic
peptidoglycan in the peptidoglycan can bind to a hyaluronic
acid.
2. The synthetic peptidoglycan of claim 1 wherein each synthetic
peptide comprises an amino acid sequence of the formula
B1-X1-X2-X3-X4-X5-X6-X7-X8-B2, wherein X8 is present or is not
present, wherein B1 is a basic amino acid, wherein B2 is a basic
amino acid, and wherein X1-X8 are non-acidic amino acids.
3. The synthetic peptidoglycan of claim 1 wherein each synthetic
peptide comprises: (i) an amino acid sequence selected from the
group consisting of: TABLE-US-00019 (SEQ ID NO: 2) GAHWQFNALTVRGG;
(SEQ ID NO: 3) GDRRRRRMWHRQ; (SEQ ID NO: 4) GKHLGGKHRRSR; (SEQ ID
NO: 5) RGTHHAQKRRS; (SEQ ID NO: 6) RRHKSGHIQGSK; (SEQ ID NO: 7)
SRMHGRVRGRHE; (SEQ ID NO: 8) RRRAGLTAGRPR; (SEQ ID NO: 9)
RYGGHRTSRKWV; (SEQ ID NO: 10) RSARYGHRRGVG; (SEQ ID NO: 11)
GLRGNRRVFARP; (SEQ ID NO: 12) SRGQRGRLGKTR; (SEQ ID NO: 13)
DRRGRSSLPKLAGPVEFPDRKIKGRR; (SEQ ID NO: 14) RMRRKGRVKHWG; (SEQ ID
NO: 15) RGGARGRHKTGR; (SEQ ID NO: 16) TGARQRGLQGGWGPRHLRGKDQPPGR;
(SEQ ID NO: 17) RQRRRDLTRVEG; (SEQ ID NO: 18)
STKDHNRGRRNVGPVSRSTLRDPIRR; (SEQ ID NO: 19) RRIGHQVGGRRN; (SEQ ID
NO: 20) RLESRAAGQRRA; (SEQ ID NO: 21) GGPRRHLGRRGH; (SEQ ID NO: 22)
VSKRGHRRTAHE; (SEQ ID NO: 23) RGTRSGSTR; (SEQ ID NO: 24)
RRRKKIQGRSKR; (SEQ ID NO: 25) RKSYGKYQGR; (SEQ ID NO: 26)
KNGRYSISR; (SEQ ID NO: 27) RRRCGQKKK; (SEQ ID NO: 28) KQKIKHVVKLK;
(SEQ ID NO: 29) KLKSQLVKRK; (SEQ ID NO: 30) RYPISRPRKR; (SEQ ID NO:
31) KVGKSPPVR; (SEQ ID NO: 32) KTFGKMKPR; (SEQ ID NO: 33)
RIKWSRVSK; and (SEQ ID NO: 34) KRTMRPTRR,
or (ii) an amino acid sequence having at least 90% sequence
identity to an amino acid sequence of (i).
4. The synthetic peptidoglycan of claim 1, wherein the glycan is
selected from the group consisting of dextran, chondroitin,
chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin,
keratin, and keratan sulfate.
5.-13. (canceled)
14. A method of treatment for arthritis in a patient, said method
comprising administering to the patient an effective amount of the
synthetic peptidoglycan of claim 1.
15. The method of claim 14 wherein each synthetic peptide comprises
an amino acid sequence of the formula
B1-X1-X2-X3-X4-X5-X6-X7-X8-B2, wherein X8 is present or is not
present, wherein B1 is a basic amino acid, wherein B2 is a basic
amino acid, and wherein X1-X8 are non-acidic amino acids.
16. The method of claim 14 wherein each synthetic peptide
comprises: (i) an amino acid sequence selected from the group
consisting of: TABLE-US-00020 (SEQ ID NO: 2) GAHWQFNALTVRGG; (SEQ
ID NO: 3) GDRRRRRMWHRQ; (SEQ ID NO: 4) GKHLGGKHRRSR; (SEQ ID NO: 5)
RGTHHAQKRRS; (SEQ ID NO: 6) RRHKSGHIQGSK; (SEQ ID NO: 7)
SRMHGRVRGRHE; (SEQ ID NO: 8) RRRAGLTAGRPR; (SEQ ID NO: 9)
RYGGHRTSRKWV; (SEQ ID NO: 10) RSARYGHRRGVG; (SEQ ID NO: 11)
GLRGNRRVFARP; (SEQ ID NO: 12) SRGQRGRLGKTR; (SEQ ID NO: 13)
DRRGRSSLPKLAGPVEFPDRKIKGRR; (SEQ ID NO: 14) RMRRKGRVKHWG; (SEQ ID
NO: 15) RGGARGRHKTGR; (SEQ ID NO: 16) TGARQRGLQGGWGPRHLRGKDQPPGR;
(SEQ ID NO: 17) RQRRRDLTRVEG; (SEQ ID NO: 18)
STKDHNRGRRNVGPVSRSTLRDPIRR; (SEQ ID NO: 19) RRIGHQVGGRRN; (SEQ ID
NO: 20) RLESRAAGQRRA; (SEQ ID NO: 21) GGPRRHLGRRGH; (SEQ ID NO: 22)
VSKRGHRRTAHE; (SEQ ID NO: 23) RGTRSGSTR; (SEQ ID NO: 24)
RRRKKIQGRSKR; (SEQ ID NO: 25) RKSYGKYQGR; (SEQ ID NO: 26)
KNGRYSISR; (SEQ ID NO: 27) RRRCGQKKK; (SEQ ID NO: 28) KQKIKHVVKLK;
(SEQ ID NO: 29) KLKSQLVKRK; (SEQ ID NO: 30) RYPISRPRKR; (SEQ ID NO:
31) KVGKSPPVR; (SEQ ID NO: 32) KTFGKMKPR; (SEQ ID NO: 33)
RIKWSRVSK; and (SEQ ID NO: 34) KRTMRPTRR,
or (ii) an amino acid sequence having at least 90% sequence
identity to an amino acid sequence of (i).
17. The method of claim 14 wherein the glycan is selected from the
group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, and keratan
sulfate.
18. The method of claim 14 wherein the synthetic peptidoglycan is
resistant to aggrecanase.
19. The method of claim 14 wherein each synthetic peptide has a
glycine-cysteine attached to the C-terminus of the peptide.
20. The method of claim 14 wherein the arthritis is selected from
the group consisting of osteoarthritis and rheumatoid
arthritis.
21. The method of claim 14 wherein the dosage of the synthetic
peptidoglycan is in a concentration ranging from about 0.1 .mu.M to
about 10 .mu.M.
22-24. (canceled)
25. The synthetic peptidoglycan of claim 1, wherein the synthetic
peptide comprises the amino acid sequence GAHWQFNALTVRGG (SEQ ID
NO: 2), or an amino acid sequence having at least about 90%
sequence identity to GAHWQFNALTVRGG (SEQ ID NO: 2).
26. The synthetic peptidoglycan of claim 1, wherein the synthetic
peptidoglycan comprises from 2 to 20 of the synthetic peptides.
27. The synthetic peptidoglycan of claim 1, wherein the synthetic
peptidoglycan comprises from 5 to 15 of the synthetic peptides.
28. The synthetic peptidoglycan of claim 1, wherein the synthetic
peptides are covalently conjugated to the glycan.
29. The synthetic peptidoglycan of claim 28, wherein the synthetic
peptides are covalently conjugated to the glycan through a
linker.
30. The The synthetic peptidoglycan of claim 1, wherein the
synthetic peptides are conjugated to backbone of the glycan.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application Ser. No. 61/489,602
filed May 24, 2011 and U.S. Provisional Application Ser. No.
61/550,621 filed Oct. 24, 2011. The disclosures of both of which
are incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention pertains to the field of hyaluronic
acid-binding synthetic peptidoglycans and methods of forming and
using the same.
BACKGROUND AND SUMMARY OF THE INVENTION
[0003] Articular cartilage is an important component for the
protection of bones in the body. In particular, articular cartilage
functions to protect articulating bones from damage by providing a
near-frictionless surface for bone movement and also providing
compressive strength to joints. Articular cartilage broadly
includes an extracellular matrix (ECM) derived from three main
components: a collagen scaffold, hyaluronic acid (HA), and
aggrecan. The material composition of articular cartilage dictates
the tissue's biological, chemical and mechanical properties. The
extracellular matrix (ECM) of healthy cartilage is primarily
composed of a network of collagen fibrils (15-22% wet weight type
II collagen), proteoglycans (4-7% wet weight), glycoproteins, water
(60-85%) and electrolytes, giving rise to a viscoelastic tissue
with depth-dependent structural and mechanical anisotropy.
[0004] Cartilage degradation and wear is a hallmark of
osteoarthritis (OA). During the initial stages of OA, aggrecan, a
major proteoglycan in cartilage, is an early component to be
degraded. The aggrecan monomer is a protein core with covalently
attached glycosaminoglycan (GAG) side chains that bind to
filamentous hyaluronic acid via globular domains and a link
protein. Proteases, such as aggrecanases, cleave aggrecan at
specific sites creating protein fragments and free GAG chains that
are unable to rebind to HA. Instead, these free GAG chains are
extruded from the matrix, which not only reduces compressive
strength, but also initiates an increase in pro-inflammatory
cytokines and matrix metalloproteases. The presence of aggrecan has
been shown to reduce diffusion of proteases protecting underlying
collagen fibers from proteolytic cleavage. Loss of aggrecan occurs
even in normal cartilage and is not immediately correlated to OA.
However, loss of type II collagen is considered an irreversible
process, leading to precocious wear.
[0005] Osteoarthritis is the most common form of arthritis,
affecting 27 million people in the US alone. The most prevalent
symptoms of osteoarthritis include immense pain, stiffening in the
joints, and tender and inflamed joints. Advanced stages of
osteoarthritis can lead to complete degradation of the articular
cartilage, causing immobility of joints and damage to the
underlying bone. The direct costs of arthritis in the United States
are estimated to be approximately $185.5 billion each year.
[0006] Although lifestyle changes and multiple medications are
often used for the treatment of osteoarthritis, there has been
little success in regeneration of defective cartilage and relieving
the symptoms caused by the loss of cartilage. This inability to
halt the progression of osteoarthritis and repair the existing
damage typically leads to an invasive, end stage cartilage
replacement procedure. Thus, an alternative treatment option for
osteoarthritis is highly desired.
[0007] The present disclosure describes improved biomaterials for
cartilage regeneration, including hyaluronic acid-binding synthetic
peptidoglycans that can be utilized to restore damaged cartilage in
an affected patient, along with methods of forming and using the
synthetic peptidoglycans. Furthermore, the hyaluronic acid-binding
synthetic peptidoglycans are designed to functionally mimic
aggrecan, resist aggrecanase degradation, and limit proteolytic
degradation. The absence of the native amino acid sequence seen in
aggrecan makes these molecules less susceptible to proteolytic
cleavage.
[0008] The following numbered embodiments are contemplated and are
non-limiting:
[0009] 1. A hyaluronic acid-binding synthetic peptidoglycan
comprising a synthetic peptide conjugated to a glycan wherein the
synthetic peptide comprises a hyaluronic acid-binding sequence.
[0010] 2. The synthetic peptidoglycan of clause 1 wherein the
synthetic peptide comprises an amino acid sequence of the formula
B1-X1-X2-X3-X4-X5-X6-X7-X8-B2, [0011] wherein X8 is present or is
not present, [0012] wherein B1 is a basic amino acid, [0013]
wherein B2 is a basic amino acid, and [0014] wherein X1-X8 are
non-acidic amino acids.
[0015] 3. The synthetic peptidoglycan of clause 1 or clause 2
wherein the synthetic peptide comprises an amino acid sequence
selected from the group consisting of:
TABLE-US-00001 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0016] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus.
[0017] 4. The synthetic peptidoglycan of any one of clauses 1 to 3
wherein the glycan is selected from the group consisting of
dextran, chondroitin, chondroitin sulfate, dermatan, dermatan
sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic
acid.
[0018] 5. The synthetic peptidoglycan of any one of clauses 1 to 4
wherein the glycan is selected from the group consisting of
chondroitin sulfate and keratan sulfate.
[0019] 6. The synthetic peptidoglycan of any one of clauses 1 to 5
wherein the synthetic peptidoglycan is resistant to
aggrecanase.
[0020] 7. The synthetic peptidoglycan of any one of clauses 1 to 6
wherein the synthetic peptidoglycan is lyophilized.
[0021] 8. A compound of formula P.sub.nG.sub.x wherein n is 1 to
20;
[0022] wherein x is 1 to 20;
[0023] wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a hyaluronic acid binding sequence; and
[0024] wherein G is a glycan. 9. A compound of formula
(P.sub.nL).sub.xG wherein n is 1 to 20;
[0025] wherein x is 1 to 20;
[0026] wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a hyaluronic acid binding sequence;
[0027] wherein L is a linker; and
[0028] wherein G is a glycan. 10. A compound of formula
P(LG.sub.n).sub.x wherein n is 1 to 20;
[0029] wherein x is 1 to 20;
[0030] wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a hyaluronic acid binding sequence;
[0031] wherein L is a linker; and
[0032] wherein G is a glycan.
[0033] 11. A compound of formula P.sub.nG.sub.x wherein n is
MWG/1000;
[0034] wherein MWG is the molecular weight of G rounded to the
nearest 1 kDa;
[0035] wherein x is 1 to 20;
[0036] wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a hyaluronic acid binding sequence; and
[0037] wherein G is a glycan.
[0038] 12. A compound of formula (P.sub.nL).sub.xG wherein n is
MWG/1000;
[0039] wherein MWG is the molecular weight of G rounded to the
nearest 1 kDa;
[0040] wherein x is 1 to 20;
[0041] wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a hyaluronic acid binding sequence;
[0042] wherein L is a linker; and
[0043] wherein G is a glycan.
[0044] 13. The compound of any one of clauses 8 to 12 wherein the
synthetic peptide comprises an amino acid sequence of the formula
B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0045] wherein X8 is present or is not present,
[0046] wherein B1 is a basic amino acid,
[0047] wherein B2 is a basic amino acid, and
[0048] wherein X1-X8 are non-acidic amino acids.
[0049] 14. The compound of any one of clauses 8 to 13 wherein the
synthetic peptide comprises an amino acid sequence selected from
the group consisting of:
TABLE-US-00002 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0050] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus.
[0051] 15. The compound of any one of clauses 8 to 14 wherein the
glycan is selected from the group consisting of dextran,
chondroitin, chondroitin sulfate, dermatan, dermatan sulfate,
heparan, heparin, keratin, keratan sulfate, and hyaluronic
acid.
[0052] 16. The compound of any one of clauses 8 to 15 wherein the
glycan is selected from the group consisting of chondroitin sulfate
and keratan sulfate.
[0053] 17. The compound of any one of clauses 8 to 16 wherein the
synthetic peptidoglycan is resistant to aggrecanase.
[0054] 18. An engineered collagen matrix comprising polymerized
collagen, hyaluronic acid, and a hyaluronic acid-binding synthetic
peptidoglycan.
[0055] 19. The engineered collagen matrix of clause 18 wherein the
collagen is selected from the group consisting of type I collagen,
type II collagen, type III collagen, type IV collagen, type IX
collagen, type XI collagen, and combinations thereof.
[0056] 20. The engineered collagen matrix of clause 18 or 19
wherein the peptide component of the synthetic peptidoglycan
comprises an amino acid sequence of the formula
B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0057] wherein X8 is present or is not present,
[0058] wherein B1 is a basic amino acid,
[0059] wherein B2 is a basic amino acid, and
[0060] wherein X1-X8 are non-acidic amino acids.
[0061] 21. The engineered collagen matrix of any one of clauses 18
to 20 wherein the peptide component of the synthetic peptidoglycan
comprises an amino acid sequence selected from the group consisting
of:
TABLE-US-00003 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0062] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus.
[0063] 22. The engineered collagen matrix of any one of clauses 18
to 21 wherein the glycan component of the synthetic peptidoglycan
is selected from the group consisting of dextran, chondroitin,
chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin,
keratin, keratan sulfate, and hyaluronic acid.
[0064] 23. The engineered collagen matrix of any one of clauses 18
to 22 wherein the glycan component of the synthetic peptidoglycan
is selected from the group consisting of chondroitin sulfate and
keratan sulfate.
[0065] 24. The engineered collagen matrix of any one of clauses 18
to 23 wherein the synthetic peptidoglycan is resistant to
aggrecanase.
[0066] 25. The engineered collagen matrix of any one of clauses 18
to 24 wherein the matrix is effective as a tissue graft.
[0067] 26. The engineered collagen matrix of clause 25 wherein the
tissue graft is implanted into a patient.
[0068] 27. The engineered collagen matrix of any one of clauses 18
to 24 wherein the matrix is in the form of a gel.
[0069] 28. The engineered collagen matrix of clause 27 wherein the
gel is administered to a patient by injection.
[0070] 29. The engineered collagen matrix of any one of clauses 18
to 24 wherein the matrix is effective as a composition for in vitro
culture of cells.
[0071] 30. The engineered collagen matrix of clause 29 wherein the
matrix further comprises an exogenous population of cells.
[0072] 31. The engineered collagen matrix of clause 30 wherein the
cells are selected from the group consisting of chondrocytes and
stem cells.
[0073] 32. The engineered collagen matrix of clause 31 wherein the
stem cells are selected from the group consisting of osteoblasts,
osteogenic cells, and mesenchymal stem cells.
[0074] 33. The engineered collagen matrix of any one of clauses 18
to 32 further comprising one or more nutrients.
[0075] 34. The engineered collagen matrix of any one of clauses 18
to 33 further comprising one or more growth factors.
[0076] 35. The engineered collagen matrix of any one of clauses 18
to 34 wherein the matrix is sterilized.
[0077] 36. A composition for in vitro culture of chondrocytes or
stem cells comprising a hyaluronic acid-binding synthetic
peptidoglycan.
[0078] 37. The composition of clause 36 wherein the peptide
component of the synthetic peptidoglycan comprises an amino acid
sequence of the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0079] wherein X8 is present or is not present,
[0080] wherein B1 is a basic amino acid,
[0081] wherein B2 is a basic amino acid, and
[0082] wherein X1-X8 are non-acidic amino acids.
[0083] 38. The composition of clause 36 or clause 37 wherein the
peptide component of the synthetic peptidoglycan comprises an amino
acid sequence selected from the group consisting of:
TABLE-US-00004 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0084] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus.
[0085] 39. The composition of any one of clauses 36 to 38 wherein
the glycan component of the synthetic peptidoglycan is selected
from the group consisting of dextran, chondroitin, chondroitin
sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin,
keratan sulfate, and hyaluronic acid.
[0086] 40. The composition of any one of clauses 36 to 39 wherein
the glycan component of the synthetic peptidoglycan is selected
from the group consisting of chondroitin sulfate and keratan
sulfate.
[0087] 41. The composition of any one of clauses 36 to 40 wherein
the synthetic peptidoglycan is resistant to aggrecanase.
[0088] 42. The composition of any one of clauses 36 to 41 wherein
the stem cells are selected from the group consisting of
osteoblasts, osteogenic cells, and mesenchymal stem cells.
[0089] 43. The composition of any one of clauses 36 to 42 further
comprising one or more nutrients.
[0090] 44. The composition of any one of clauses 36 to 43 further
comprising one or more growth factors.
[0091] 45. The composition of any one of clauses 36 to 44 wherein
the composition is sterilized.
[0092] 46. An additive for a biomaterial cartilage or bone
replacement composition comprising a hyaluronic acid-binding
synthetic peptidoglycan for addition to an existing biomaterial
cartilage or bone replacement material.
[0093] 47. The additive of clause 46 wherein the peptide component
of the synthetic peptidoglycan comprises an amino acid sequence of
the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0094] wherein X8 is present or is not present,
[0095] wherein B1 is a basic amino acid,
[0096] wherein B2 is a basic amino acid, and
[0097] wherein X1-X8 are non-acidic amino acids.
[0098] 48. The additive of clause 46 or clause 47 wherein the
peptide component of the synthetic peptidoglycan comprises an amino
acid sequence selected from the group consisting of:
TABLE-US-00005 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0099] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus.
[0100] 49. The additive of any one of clauses 46 to 48 wherein the
glycan component of the synthetic peptidoglycan is selected from
the group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, keratan
sulfate, and hyaluronic acid.
[0101] 50. The additive of any one of clauses 46 to 49 wherein the
glycan is selected from the group consisting of chondroitin sulfate
and keratan sulfate.
[0102] 51. The additive of any one of clauses 46 to 50 wherein the
synthetic peptidoglycan is resistant to aggrecanase.
[0103] 52. A method of treatment for arthritis in a patient, said
method comprising the step of administering to the patient a
hyaluronic acid-binding synthetic peptidoglycan, wherein the
synthetic peptidoglycan reduces a symptom associated with the
arthritis.
[0104] 53. The method of clause 52 wherein the peptide component of
the synthetic peptidoglycan comprises an amino acid sequence of the
formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0105] wherein X8 is present or is not present,
[0106] wherein B1 is a basic amino acid,
[0107] wherein B2 is a basic amino acid, and
[0108] wherein X1-X8 are non-acidic amino acids.
[0109] 54. The method of clause 52 or clause 53 wherein the peptide
component of the synthetic peptidoglycan comprises an amino acid
sequence selected from the group consisting of:
TABLE-US-00006 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0110] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus.
[0111] 55. The method of any one of clauses 52 to 54 wherein the
glycan component of the synthetic peptidoglycan is selected from
the group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, keratan
sulfate, and hyaluronic acid.
[0112] 56. The method of any one of clauses 52 to 55 wherein the
glycan is selected from the group consisting of chondroitin sulfate
and keratan sulfate.
[0113] 57. The method of any one of clauses 52 to 56 wherein the
synthetic peptidoglycan is resistant to aggrccanasc.
[0114] 58. The method of any one of clauses 52 to 57 wherein the
arthritis is osteoarthritis.
[0115] 59. The method of any one of clauses 52 to 57 wherein the
arthritis is rheumatoid arthritis.
[0116] 60. The method of any one of clauses 52 to 59 wherein the
synthetic peptidoglycan is administered to the patient by
injection.
[0117] 61. The method of clause 60 wherein the injection is an
intraarticular injection.
[0118] 62. The method of clause 60 wherein the injection is into a
joint capsule of the patient.
[0119] 63. The method of any one of clauses 52 to 62 wherein the
synthetic peptidoglycan is administered using a needle or a device
for infusion.
[0120] 64. The method of any one of clauses 52 to 63 wherein the
synthetic peptidoglycan acts as a lubricant.
[0121] 65. The method of any one of clauses 52 to 64 wherein the
synthetic peptidoglycan prevents bone on bone articulation or
prevents cartilage loss.
[0122] 66. A method of preparing a biomaterial or bone cartilage
replacement, said method comprising the step of combining the
synthetic peptidoglycan and an existing biomaterial or bone
cartilage replacement material.
[0123] 67. The method of clause 66 wherein the peptide component of
the synthetic peptidoglycan comprises an amino acid sequence of the
formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0124] wherein X8 is present or is not present,
[0125] wherein B1 is a basic amino acid,
[0126] wherein B2 is a basic amino acid, and
[0127] wherein X1-X8 are non-acidic amino acids.
[0128] 68. The method of clause 66 or clause 67 wherein the peptide
component of the synthetic peptidoglycan comprises an amino acid
sequence selected from the group consisting of:
TABLE-US-00007 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKTKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0129] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus.
[0130] 69. The method of any one of clauses 66 to 68 wherein the
glycan component of the synthetic peptidoglycan is selected from
the group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, keratan
sulfate, and hyaluronic acid.
[0131] 70. The method of any one of clauses 66 to 69 wherein the
glycan is selected from the group consisting of chondroitin sulfate
and keratan sulfate.
[0132] 71. The method of any one of clauses 66 to 70 wherein the
synthetic peptidoglycan is resistant to aggrecanase.
[0133] 72. A method of reducing or preventing hyaluronic acid
degradation in a patient, said method comprising administering to
the patient a hyaluronic acid-binding synthetic peptidoglycan.
[0134] 73. The method of clause 72 wherein the peptide component of
the synthetic peptidoglycan comprises an amino acid sequence of the
formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0135] wherein X8 is present or is not present,
[0136] wherein B1 is a basic amino acid,
[0137] wherein B2 is a basic amino acid, and
[0138] wherein X1-X8 are non-acidic amino acids.
[0139] 74. The method of clause 72 or clause 73 wherein the peptide
component of the synthetic peptidoglycan comprises an amino acid
sequence selected from the group consisting of:
TABLE-US-00008 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0140] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus
[0141] 75. The method of any one of clauses 72 to 74 wherein the
glycan component of the synthetic peptidoglycan is selected from
the group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, keratan
sulfate, and hyaluronic acid.
[0142] 76. The method of any one of clauses 72 to 75 wherein the
glycan is selected from the group consisting of chondroitin sulfate
and keratan sulfate.
[0143] 77. The method of any one of clauses 72 to 76 wherein the
synthetic peptidoglycan is resistant to aggrecanase.
[0144] 78. The method of any one of clauses 72 to 77 wherein the
synthetic peptidoglycan is administered to the patient by
injection.
[0145] 79. The method of clause 78 wherein the injection is an
intraarticular injection.
[0146] 80. The method of clause 78 wherein the injection is into a
joint capsule of the patient.
[0147] 81. The method of any one of clauses 72 to 80 wherein the
rate of hyaluronic acid degradation is reduced.
[0148] 82. A method for correcting or modifying a tissue defect in
a patient comprising
[0149] administering into the tissue defect a hyaluronic
acid-binding synthetic peptidoglycan wherein the defect is
corrected or modified.
[0150] 83. The method of clause 82 wherein the peptide component of
the synthetic peptidoglycan comprises an amino acid sequence of the
formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0151] wherein X8 is present or is not present,
[0152] wherein B1 is a basic amino acid,
[0153] wherein B2 is a basic amino acid, and
[0154] wherein X1-X8 are non-acidic amino acids.
[0155] 84. The method of clause 82 or clause 83 wherein the peptide
component of the synthetic peptidoglycan comprises an amino acid
sequence selected from the group consisting of:
TABLE-US-00009 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0156] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus.
[0157] 85. The method of any one of clauses 82 to 84 wherein the
glycan component of the synthetic peptidoglycan is selected from
the group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, keratan
sulfate, and hyaluronic acid.
[0158] 86. The method of any one of clauses 82 to 85 wherein the
glycan is selected from the group consisting of chondroitin sulfate
and keratan sulfate.
[0159] 87. The method of any one of clauses 82 to 86 wherein the
synthetic peptidoglycan is resistant to aggrecanase.
[0160] 88. The method of any one of clauses 82 to 87 wherein the
synthetic peptidoglycan is administered to the patient by
injection.
[0161] 89. The method of clause 88 wherein the injection is
subcutaneous.
[0162] 90. The method of any one of clauses 82 to 89 wherein the
defect is a cosmetic defect.
[0163] 91. A dermal filler comprising a hyaluronic acid-binding
synthetic peptidoglycan.
[0164] 92. The dermal filler of clause 91 wherein the peptide
component of the synthetic peptidoglycan comprises an amino acid
sequence of the formula B1-X1-X2-X3 -X4-X5 -X6-X7-X8-B2,
[0165] wherein X8 is present or is not present,
[0166] wherein B1 is a basic amino acid,
[0167] wherein B2 is a basic amino acid, and
[0168] wherein X1-X8 are non-acidic amino acids.
[0169] 93. The dermal filler of clause 91 or clause 92 wherein the
peptide component of the synthetic peptidoglycan comprises an amino
acid sequence selected from the group consisting of:
TABLE-US-00010 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0170] In each of the above peptide embodiments, the peptide may
have a glycine-cysteine (GC) attached to the C-terminus of the
peptide, or a glycine-cysteine-glycine (GCG) attached to the
N-terminus.
[0171] 94. The dermal filler of any one of clauses 91 to 93 further
comprising hyaluronic acid.
[0172] 95. A method of reducing or preventing collagen degradation,
said method comprising the steps of
[0173] contacting a hyaluronic acid-binding synthetic peptidoglycan
with hyaluronic acid in the presence of collagen, and
[0174] reducing or preventing collagen degradation.
[0175] 96. The method of clause 95 wherein the peptide component of
the synthetic peptidoglycan comprises an amino acid sequence of the
formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0176] wherein X8 is present or is not present,
[0177] wherein B1 is a basic amino acid,
[0178] wherein B2 is a basic amino acid, and
[0179] wherein X1-X8 are non-acidic amino acids.
[0180] 97. The method of clause 95 or clause 96 wherein the peptide
component of the synthetic peptidoglycan comprises an amino acid
sequence selected from the group consisting of:
TABLE-US-00011 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0181] 98. The method of any one of clauses 95 to 97 wherein the
glycan component of the synthetic peptidoglycan is selected from
the group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, keratan
sulfate, and hyaluronic acid.
[0182] 99. The method of any one of clauses 95 to 98 wherein the
glycan is selected from the group consisting of chondroitin sulfate
and keratan sulfate.
[0183] 100. The method of any one of clauses 95 to 99 wherein the
synthetic peptidoglycan is resistant to aggrecanase.
[0184] 101. The method of any one of clauses 95 to 100 wherein the
rate of hyaluronic acid degradation is reduced.
[0185] 102. A method of increasing pore size in an engineered
collagen matrix, said method comprising the steps of
[0186] combining collagen, hyaluronic acid, and a hyaluronic
acid-binding synthetic peptidoglycan, and
[0187] increasing the pore size in the matrix.
[0188] 103. The method of clause 102 wherein the peptide component
of the synthetic peptidoglycan comprises an amino acid sequence of
the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0189] wherein X8 is present or is not present,
[0190] wherein B1 is a basic amino acid,
[0191] wherein B2 is a basic amino acid, and
[0192] wherein X1-X8 are non-acidic amino acids.
[0193] 104. The method of clause 102 or clause 103 wherein the
peptide component of the synthetic peptidoglycan comprises an amino
acid sequence selected from the group consisting of:
TABLE-US-00012 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0194] 105. The method of any one of clauses 102 to 104 wherein the
glycan component of the synthetic peptidoglycan is selected from
the group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, keratan
sulfate, and hyaluronic acid.
[0195] 106. The method of any one of clauses 102 to 105 wherein the
glycan is selected from the group consisting of chondroitin sulfate
and keratan sulfate.
[0196] 107. The method of any one of clauses 102 to 106 wherein the
synthetic peptidoglycan is resistant to aggrecanase.
[0197] 108. The method of any one of clauses 102 to 107 wherein the
matrix is sterilized.
[0198] 109. The method of any one of clauses 102 to 108 wherein the
matrix further comprises chondrocytes or stem cells.
[0199] 110. The method of clause 109 wherein the stem cells are
selected from the group consisting of osteoblasts, osteogenic
cells, and mesenchymal stem cells.
[0200] 111. The method of any one of clauses 102 to 110 wherein the
matrix further comprises one or more nutrients.
[0201] 112. The method of any one of clauses 102 to 111 wherein the
matrix further comprises one or more growth factors.
[0202] 113. A method of reducing or preventing chondroitin sulfate
degradation, said method comprising the steps of
[0203] contacting a hyaluronic acid-binding synthetic peptidoglycan
with hyaluronic acid in the presence of collagen, and
[0204] reducing or preventing chondroitin sulfate degradation.
[0205] 114. The method of clause 113 wherein the peptide component
of the synthetic peptidoglycan comprises an amino acid sequence of
the formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0206] wherein X8 is present or is not present,
[0207] wherein B1 is a basic amino acid,
[0208] wherein B2 is a basic amino acid, and
[0209] wherein X1-X8 are non-acidic amino acids.
[0210] 115. The method of clause 113 or clause 114 wherein the
peptide component of the synthetic peptidoglycan comprises an amino
acid sequence selected from the group consisting of:
TABLE-US-00013 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
[0211] 116. The method of any one of clauses 113 to 115 wherein the
glycan component of the synthetic peptidoglycan is selected from
the group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, keratan
sulfate, and hyaluronic acid.
[0212] 117. The method of any one of clauses 113 to 116 wherein the
glycan is selected from the group consisting of chondroitin sulfate
and keratan sulfate.
[0213] 118. The method of any one of clauses 113 to 117 wherein the
synthetic peptidoglycan is resistant to aggrecanase.
[0214] 119. The method of any one of clauses 113 to 118 wherein the
rate of chondroitin sulfate degradation is reduced.
[0215] 120. The synthetic peptidoglycan, compound, engineered
collagen matrix, composition, additive, method, or dermal filler of
any of the preceding clauses wherein the peptide component of the
synthetic peptidoglycan has a glycine-cysteine (GC) attached to the
C-terminus of the peptide.
[0216] 121. The synthetic peptidoglycan, compound, engineered
collagen matrix, composition, additive, method, or dermal filler of
any of the preceding clauses wherein the peptide component of the
synthetic peptidoglycan has a glycine-cysteine-glycine (GCG)
attached to the N-terminus of the peptide. p 122. The synthetic
peptidoglycan, compound, engineered collagen matrix, composition,
additive, method, or dermal filler of any of the preceding clauses
wherein the synthetic peptidoglycan is resistant to matrix metallo
proteases.
[0217] 123. The synthetic peptidoglycan, compound, engineered
collagen matrix, composition, additive, method, or dermal filler of
clause 122 wherein the matrix metallo protease is aggrecanase.
[0218] 124. The synthetic peptidoglycan, compound, engineered
collagen matrix, composition, additive, method, or dermal filler of
any of the preceding clauses wherein the dosage of the synthetic
peptidoglycan is in a concentration ranging from about 0.01 uM to
about 100 uM.
[0219] 125. The synthetic peptidoglycan, compound, engineered
collagen matrix, composition, additive, method, or dermal filler of
any of the preceding clauses wherein the dosage of the synthetic
peptidoglycan is in a concentration ranging from about 0.1 uM to
about 10 uM.
BRIEF DESCRIPTION OF THE DRAWINGS
[0220] FIG. 1 shows a reaction schematic for the production of an
embodiment of the hyaluronic acid-binding synthetic peptidoglycan.
Reaction steps are detailed in bold font.
[0221] FIG. 2 shows a standard curve of
N-[.beta.-Maleimidopropionic acid]hydrazide, trifluoroacetic acid
salt (hereinafter "BMPH") absorbance (215 nm) based on amount (mg)
of BMPH injected. The standard curve was used to determine the
amount of BMPH consumed during the coupling reaction.
[0222] FIG. 3 shows binding of the hyaluronic acid-binding
synthetic peptidoglycan to the immobilized hyaluronic acid (HA).
Nine HA-binding peptides (e.g., GAHWQFNALTVRGGGC; hereinafter "GAH"
or "mAGC") were attached to the functionalized glycosaminoglycan
(e.g., chondroitin sulfate, hereinafter "CS") backbone.
Concentrations of synthetic peptidoglycans were increased from 0.01
.mu.M to 100 .mu.M.
[0223] FIG. 4 shows HA binding of the synthetic peptidoglycan as
determined by rheo logical frequency sweep (Panel A). The storage
modulus of the HA mixtures was analyzed at an oscillatory frequency
of 5.012 Hz. At this frequency, a noticeable load was provided
while the integrity of the HA chains was maintained. Statistical
analysis (.alpha.=0.05) showed that HA+CS and HA were significantly
different (denoted *), and that HA+10.5 GAH-CS and HA+CS were
significantly different (denoted **). Panel B is an alternative
representation of the same data shown in Panel A.
[0224] FIG. 5 shows quatification of the turbidity of the collagen
type I plus treatment groups during collagen fibril formation. The
absorbance at 313 nm was measured every 3 minutes. After one hour
(i.e., timepont number 20), all treatment groups had completely
formed networks. No significant differences (.alpha.=0.05) existed
between treatment groups with respect to the maximum absorbance or
the time to half maximum absorbance.
[0225] FIG. 6 shows the compressive engineering stress withstood by
the collagen gels based on an applied engineering strain of 1% per
second. Statistical analysis (.alpha.=0.05) demonstrated that the
addition of 10.5 GAH-CS resulted in a significant difference in
peak engineering stress, in addition to the engineering stress
analyzed at engineering strains of 5%, 7.5%, and 10%.
[0226] FIG. 7 shows the storage modulus of collagen mixtures
measured at an oscillatory frequency of 0.5012 Hz. Statistical
analysis (.alpha.=0.05) demonstrated that the addition of 10.5
GAH-CS resulted in a significant increase in the storage modulus of
the collagen gel (denoted *).
[0227] FIG. 8 shows the percent degradation of HA mixtures due to
the addition of hyaluronidase to the mixtures (Panel A). The
percent degradation was determined by the changes in the dynamic
viscosity of the HA mixtures. Dynamic viscosity measurements were
initially taken of the mixtures, and served as the baseline from
which the percent degradations were calculated. The 0 hour
timepoint was taken after the addition of hyaluronidase, the
sufficient mixing of the samples, and the pipetting onto the
rheometer stage, and approximately 2 minutes passed between the
addition of the hyaluronidase and the measurement of the dynamic
viscosity. Statistical analysis demonstrated significant
differences (.alpha.=0.05) in the percent degradation for the 10.5
GAH-CS sample at both the 0 hour and 2 hour timepoints. Panel B
shows the same data represented as normalized dynamic viscosity
(mean.+-.SE, n=3) of HA mixtures due to the addition of
hyaluronidase. Dynamic viscosity measurements were initially taken
of the mixtures before hyaluronidase was added, and these values
served as the baseline from which the normalized dynamic
viscosities were calculated. The normalized dynamic viscosities
were determined by taking each measured dynamic viscosity after the
addition of hyaluronidase and dividing this value by the initial
dynamic viscosity of that sample. Statistical analysis
(.alpha.=0.05) was conducted, and significant differences were seen
in the normalized degradation for the 10.5 GAH-CS sample at both
the 0 hr and 2 hr timepoints.
[0228] FIG. 9 shows representative cryo-SEM images (10,000.times.
magnification with 5 .mu.m scale bar) of the CI scaffold associated
with each cartilage ECM replicate. Panel A represents the CI
control. Panel B represents CI+HA+CS. Panel C represents CI+HA+10.5
GAH-CS.
[0229] FIG. 10 shows the percent degradation (mean.+-.SE, n=3) of
CI in ECM replicates exposed to MMP-I throughout a 50 hr duration.
Statistical analysis (p<0.05) of the different treatments
revealed that all three treatments (CI control, CI+HA+CS, and
CI+HA+10.5 GAH-CS) were significantly different from each
other.
[0230] FIG. 11 shows the cumulative chondroitin sulfate (CS) loss
over an eight-day culture period in media stimulated with and
without IL-1.beta.. CS loss was measured by a DMMB assay. The
addition of mAGC had a significant effect on loss of CS from the
scaffolds (p<0.001). ** denotes statistical significance between
scaffold prepared without aggrecan mimic and those prepared with
mAGC. + denotes statistical significance between scaffold treated
with and without IL-1.beta. (p<0.05). Bars represent average
.+-.SEM (n=3).
[0231] FIG. 12 shows the cumulative collagen breakdown over an
eight-day culture period in media stimulated with and without
IL-1.beta.. Collagen breakdown was measured by a Sircol assay. The
addition of aggrecan mimic had a significant effect on loss of
collagen from the scaffolds (p<0.02). ** denotes statistical
significance between scaffold prepared without aggrecan mimic and
those prepared with mAGC. + denotes statistical significance
between scaffold treated with and without IL-1.beta. (p<0.05).
Bars represent average .+-.SEM (n=3).
[0232] FIG. 13 represents a platform to study the efficacy of the
peptidoglycan ex vivo. 0.5% trypsin was used to remove native
aggrecan from bovine cartilage explants. Removal of aggrecan was
confirmed by DMMB assay. Graphs represent the amount of aggrecan
removed compared to positive control.
[0233] FIG. 14 shows an assay to monitor peptidoglycan diffusion
through the cartilage matrix. The Y-axis represents the difference
in DMMB assay absorbance values read from aggrecan-depleted
cartilage plugs treated with/without peptidoglycan. The X-axis
represents the distance from the articular surface of cartilage to
subchondral bone. Bars represent average difference .+-.SEM
(n=3).
[0234] FIG. 15 shows Safranin O and Avidin-Biotin stains of bovine
cartilage explants. A midsagittal cut was made through the matrix
and probed for residual aggrecan (top panel, dark staining) and
biotin (bottom panel, dark staining) respectively. Collagen type II
binding peptidoglycan [WYRGRLGC; "mAG(II)C"] was diffused through
the explant. Higher magnification (20.times.m) of this tissue slice
indicated that mAG(II)C penetrates approximately 200 um into
tissue.
[0235] FIG. 16 shows Avidin-Biotin stains of cartilage explants.
Peptidoglycans (mAG(II)C and mAGC) were diffused through the
cartilage explant. Images indicate depth of penetration of each
(dark staining).
[0236] FIG. 17 shows that the addition of peptidoglycans in
aggrecan depleted (AD) explants increased compressive stiffness.
Addition of the HA binding peptidoglycan (mAGC) significantly
restored stiffness of cartilage explants to a higher extent as
compared to the collagen type TI binding peptidoglycan (mAG(TI)C).
Significance, denoted as *, specified an increase in compressive
stiffness between AD and AD+mACG augmented explants (p<0.005).
Data is presented as mean.+-.SEM (n=5).
[0237] FIG. 18 (A) shows a schematic representation of probe bound
to MMP-13. BHQ-3 black hole quencher 3 and CY5.5 absorbed and
emitted at 695 nm respectively. Arrow and italics indicate the
cleavage site. (B) shows the concentration profile of probe
activity with and without MMP-13: Left, fluorescence imaging
sections of 96-well microplate; Right, recovery of fluorescence
emission intensity (695 nm).
[0238] FIG. 19 shows the extent of inflammation indicated by the
MMP-13 probe in Sprague-Dawley rats treated with and without
peptidoglycan at four, six and eight weeks post surgery.
[0239] FIG. 20 shows a x-ray images of Sprague-Dawley rat knee
joints showing injured knee 6 weeks and 8 weeks following OA
induction (Panels A and D, respectively), injured knee with
peptidoglycan treatment (Panels B and E, respectively), and normal
knee (Panel C) six weeks after osteoarthritis induction
surgery.
[0240] FIG. 21 shows microCT of Sprague-Dawley rats indicating
re-growth of new cartilage six and eight weeks after OA induction
surgery. Injured knees 6 weeks and 8 weeks following OA induction
are shown in Panels A and D, respectively. Injured knees following
peptidoglycan treatment are shown in Panels B and E, respectively.
Normal knee is shown in panel C.
[0241] FIG. 22 shows that the addition of mAGC to collagen
scaffolds increased the storage modulus and compressive stiffness.
Frequency sweeps (A) on collagen scaffolds indicated an increase in
storage modulus over a range of 0.1-2.0 Hz. Similarly, compressive
stiffness (B) showed an increase in values when the scaffold was
prepared with the addition of mAGC. Significance is denoted as *
(p<0.0001). Data is presented as mean .+-.SEM (n=5).
[0242] FIG. 23 shows cumulative chondroitin sulfate (CS) loss over
an eight-day culture period in media stimulated with and without
IL-1.beta.. CS loss was measured by a DMMB assay. Scaffold
compositions (A-H) are described in Table 3. The addition of mAGC
had a significant effect on loss of CS from the scaffolds
(p<0.001). * denotes statistical significance between scaffold A
and C, and scaffold E and G. (p<0.05). Bars represent average
.+-.SEM (n=3).
[0243] FIG. 24 shows cumulative collagen breakdown over an
eight-day culture period in media stimulated with and without
1L-1.beta.. Collagen breakdown was measured by a Sircol assay.
Scaffold compositions (A-H) are described in Table 3. The addition
of aggrecan mimic had a significant effect on loss of collagen from
the scaffolds (p<0.02). * denotes statistical significance
between scaffold A and C, and scaffold E and G. (p<0.05). Bars
represent average .+-.SEM (n=3).
[0244] FIG. 25 shows real-time PCR analysis for aggrecan and
collagen type II expressed by bovine chondrocytes cultured in
unaligned (A) and aligned (B) collagen scaffolds. Values were
normalized to endogenous GAPDH expression. Addition of mAGC
statistically changed aggrecan and collagen type II expression
(p.sub.aggrecan<0.02 and p.sub.collagen<0.001) respectively.
There was also a statistical difference in aggrecan and collagen
type II expression between unaligned and aligned scaffolds
(p<0.001). Similarly, the aggrecan and collagen type II
expression differed between scaffolds treated with and without
IL-1.beta. (p<0.01). Scaffold compositions (A-H) are described
in Table 3. Bars represent average .+-.SEM (n=4).
DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS
[0245] As used herein, a "hyaluronic acid-binding synthetic
peptidoglycan" means a synthetic peptide conjugated to a glycan
where the peptide comprises a hyaluronic acid-binding sequence.
Various embodiments of the invention are described herein as
follows. In one embodiment described herein, a hyaluronic
acid-binding synthetic peptidoglycan is provided. The hyaluronic
acid-binding synthetic peptidoglycan comprises a synthetic peptide
conjugated to a glycan wherein the synthetic peptide comprises a
hyaluronic acid-binding sequence.
[0246] In another embodiment, a compound of the formula
P.sub.nG.sub.x is described wherein n is 1 to 20; wherein x is 1 to
20; wherein P is a synthetic peptide of about 5 to about 40 amino
acids comprising a hyaluronic acid binding sequence; and wherein G
is a glycan.
[0247] In yet another embodiment, a compound of the formula
(P.sub.nL) .sub.xG is described
[0248] wherein n is 1 to 20;
[0249] wherein x is 1 to 20;
[0250] wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a hyaluronic acid binding sequence;
[0251] wherein L is a linker; and
[0252] wherein G is a glycan.
[0253] In another embodiment, a compound of the formula
P(LG.sub.n).sub.x is described
[0254] wherein n is 1 to 20;
[0255] wherein x is 1 to 20;
[0256] wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a hyaluronic acid binding sequence;
[0257] wherein L is a linker; and wherein G is a glycan.
[0258] In yet another embodiment, a compound of the formula
P.sub.nG.sub.x is described
[0259] wherein n is MWG/1000;
[0260] wherein MWG is the molecular weight of G rounded to the
nearest 1 kDa;
[0261] wherein x is 1 to 20;
[0262] wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a hyaluronic acid binding sequence; and
[0263] wherein G is a glycan.
[0264] In another embodiment, a compound of the formula
(P.sub.nL).sub.xG is described
[0265] wherein n is MWG/1000;
[0266] wherein MWG is the molecular weight of G rounded to the
nearest 1 kDa;
[0267] wherein x is 1 to 20;
[0268] wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a hyaluronic acid binding sequence;
[0269] wherein L is a linker; and
[0270] wherein G is a glycan.
[0271] For purposes of this disclosure, the hyaluronic acid-binding
synthetic peptidoglycans and compounds described in the preceding
paragraphs are collectively referred to as "hyaluronic acid-binding
synthetic peptidoglycans" or "synthetic peptidoglycans."
[0272] In each of the above peptide embodiments, the synthetic
peptidoglycan may comprise 5-15 peptide molecules (n is 5-15), 5-20
peptide molecules (n is 5-20), 1-20 peptide molecules (n is 1-20),
or 1-25 peptide molecules (n is 1-25). In one embodiment, n is
selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25
peptide molecules.
[0273] In another illustrative embodiment described herein, an
engineered collagen matrix is provided. The matrix comprises
polymerized collagen, hyaluronic acid, and a hyaluronic
acid-binding synthetic peptidoglycan. In another embodiment, a
composition for in vitro culture of chondrocytes or stem cells is
provided. The composition comprises any of the hyaluronic
acid-binding synthetic peptidoglycans described in this
disclosure.
[0274] In another embodiment described herein, a method of
increasing pore size in an engineered collagen matrix is provided.
The method comprises the steps of combining collagen, hyaluronic
acid, and a hyaluronic acid-binding synthetic peptidoglycan and
increasing the pore size in the matrix.
[0275] In yet another illustrative embodiment, a method of
decreasing cartilage wear or erosion in a patient is provided. The
method comprises the step of administering to the patient a
hyaluronic acid-binding synthetic peptidoglycan, wherein the
synthetic peptidoglycan decreases wear or erosion of the cartilage.
In one embodiment, the cartilage erosion or wear may be caused by
arthritis. In one embodiment, the cartilage erosion or wear may be
caused by aging, obesity, trauma or injury, an anatomic
abnormality, genetic diseases, metabolic imbalances, inflammation,
or the like.
[0276] In yet another illustrative embodiment, a method of
treatment for arthritis in a patient is provided. The method
comprises the step of administering to the patient a hyaluronic
acid-binding synthetic peptidoglycan, wherein the synthetic
peptidoglycan reduces a symptom associated with the arthritis.
[0277] In another illustrative embodiment, a method of reducing or
preventing hyaluronic acid degradation in a patient is provided.
The method comprises administering to the patient a hyaluronic
acid-binding synthetic peptidoglycan.
[0278] In another illustrative embodiment, a method of reducing or
preventing collagen degradation is provided. The method comprises
the steps of contacting a hyaluronic acid-binding synthetic
peptidoglycan with hyaluronic acid in the presence of collagen, and
reducing or preventing collagen degradation.
[0279] In yet another illustrative embodiment, a method for
correcting or modifying a tissue defect in a patient is provided.
The method comprises administering into the tissue defect a
hyaluronic acid-binding synthetic peptidoglycan wherein the defect
is corrected or modified.
[0280] In another illustrative embodiment described herein, a
dermal filler is provided. The filler comprises a hyaluronic
acid-binding synthetic peptidoglycan. In one embodiment, the filler
further comprises hyaluronic acid.
[0281] In yet another embodiment, an additive for a biomaterial
cartilage or bone replacement composition is provided. The additive
comprises a hyaluronic acid-binding synthetic peptidoglycan for
addition to an existing biomaterial cartilage or bone replacement
material. In another embodiment described herein, a method of
preparing a biomaterial or bone cartilage replacement is provided.
The method comprises the step of combining the synthetic
peptidoglycan and an existing biomaterial or bone cartilage
replacement material.
[0282] In the various embodiments, the peptide component of the
synthetic peptidoglycan comprises an amino acid sequence of the
formula B1-X1-X2-X3-X4-X5-X6-X7-X8-B2,
[0283] wherein X8 is present or is not present,
[0284] wherein B1 is a basic amino acid,
[0285] wherein B2 is a basic amino acid, and
[0286] wherein X1-X8 are non-acidic amino acids.
[0287] In another embodiment, the peptide component of the
synthetic peptidoglycan can comprise or can be an amino acid
sequence of the formula B1-X1-B2-X2-X3-X4-X5-X6-X7-X8-X9-B3,
[0288] wherein X9 is present or is not present,
[0289] wherein B1 is a basic amino acid,
[0290] wherein B2 is a basic amino acid,
[0291] wherein B3 is a basic amino acid, and
[0292] wherein X1-X9 are non-acidic amino acids.
[0293] In another embodiment, the synthetic peptide can comprise or
can be an amino acid sequence of the formula
B1-X1-X2-X3-X4-X5-X6-X7-X8-B2-X9-B3,
[0294] wherein X8 is present or is not present,
[0295] wherein B1 is a basic amino acid,
[0296] wherein B2 is a basic amino acid,
[0297] wherein B3 is a basic amino acid, and
[0298] wherein X1-X9 are non-acidic amino acids.
[0299] As used herein, a "basic amino acid" is selected from the
group consisting of lysine, arginine, or histidine. As used herein,
a "non-acidic amino acid" is selected from the group consisting of
alanine, arginine, asparagine, cysteine, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, and valine.
[0300] In the various illustrative embodiments described herein,
the peptide component of the synthetic peptidoglycan can comprise
an amino acid sequence selected from the group consisting of:
TABLE-US-00014 GAHWQFNALTVRGG; GDRRRRRMWHRQ; GKHLGGKHRRSR;
RGTHHAQKRRS; RRHKSGHIQGSK; SRMHGRVRGRHE; RRRAGLTAGRPR;
RYGGHRTSRKWV; RSARYGHRRGVG; GLRGNRRVFARP; SRGQRGRLGKTR;
DRRGRSSLPKLAGPVEFPDRKIKGRR; RMRRKGRVKHWG; RGGARGRHKTGR;
TGARQRGLQGGWGPRHLRGKDQPPGR; RQRRRDLTRVEG;
STKDHNRGRRNVGPVSRSTLRDPIRR; RRIGHQVGGRRN; RLESRAAGQRRA;
GGPRRHLGRRGH; VSKRGHRRTAHE; RGTRSGSTR; RRRKKIQGRSKR; RKSYGKYQGR;
KNGRYSISR; RRRCGQKKK; KQKIKHVVKLK; KLKSQLVKRK; RYPISRPRKR;
KVGKSPPVR; KTFGKMKPR; RIKWSRVSK; and KRTMRPTRR.
In each of the above peptide embodiments, the peptide may have a
glycine-cysteine attached to the C-terminus of the peptide, and/or
a glycine-cysteine-glycine (GCG) attached to the N-terminus of the
peptide. In various embodiments described herein, the peptide
component of the synthetic peptidoglycan comprises any amino acid
sequence described in the preceding paragraph or an amino acid
sequence with 80%, 85%, 90%, 95%, 98%, or 100% homology to any of
these amino acid sequences.
[0301] Additional peptides that can be included as the peptide
component of the hyaluronic acid-binding synthetic peptidoglycans
include peptides described in Amemiya et al., Biochem. Biophys.
Acta, vol. 1724, pp. 94-99 (2005), incorporated herein by
reference. These peptides have an Arg-Arg motif and include
peptides selected from the group consisting of:
TABLE-US-00015 RRASRSRGQVGL; GRGTHHAQKRRS; QPVRRLGTPVVG;
ARRAEGKTRMLQ; PKVRGRRHQASG; SDRHRRRREADG; NQRVRRVKHPPG;
RERRERHAVARHGPGLERDARNLARR; TVRPGGKRGGQVGPPAGVLHGRRARS;
NVRSRRGHRMNS; DRRRGRTRNIGN; KTAGHGRRWSRN; AKRGEGRREWPR;
GGDRRKAHKLQA; RRGGRKWGSFEG; and RQRRRDLTRVEG.
In each of the above peptide embodiments, the peptide may have a
glycine-cysteine attached to the C-terminus of the peptide. In each
of the above peptide embodiments, the peptide may have a
glycine-cysteine-glycine (GCG) attached to the N-terminus of the
peptide. In various embodiments described herein, the peptide
component of the synthetic peptidoglycan comprises any amino acid
sequence described in the preceding paragraph or an amino acid
sequence with 80%, 85%, 90%, 95%, 98%, or 100% homology to any of
these amino acid sequences.
[0302] In other embodiments, peptides described in Yang et al.,
EMBO Journal, vol. 13, pp. 286-296 (1994), incorporated herein by
reference, and Goetinck et al., J. Cell. Biol., vol. 105, pp.
2403-2408 (1987), incorporated herein by reference, can be used in
the hyaluronic acid-binding synthetic peptidoglycans described
herein including peptides selected from the group consisting of
RDGTRYVQKGEYR, HREARSGKYK, PDKKHKLYGV, and WDKERSRYDV. In each of
these embodiments, the peptide may have a glycine-cysteine attached
to the C-terminus of the peptide. In each of these embodiments, the
peptide may have a glycine-cysteine-glycine (GCG) attached to the
N-terminus of the peptide. In other embodiments, the peptide
component of the synthetic peptidoglycan comprises an amino acid
sequence with 80%, 85%, 90%, 95%, 98%, or 100% homology to any of
these amino acid sequences.
[0303] In various embodiments, the peptide component of the
synthetic peptidoglycan described herein can be modified by the
inclusion of one or more conservative amino acid substitutions. As
is well-known to those skilled in the art, altering any
non-critical amino acid of a peptide by conservative substitution
should not significantly alter the activity of that peptide because
the side-chain of the replacement amino acid should be able to form
similar bonds and contacts to the side chain of the amino acid
which has been replaced. Non-conservative substitutions are
possible provided that these do not excessively affect the
hyaluronic acid binding activity of the peptide.
[0304] As is well-known in the art, a "conservative substitution"
of an amino acid or a "conservative substitution variant" of a
peptide refers to an amino acid substitution which maintains: 1)
the secondary structure of the peptide; 2) the charge or
hydrophobicity of the amino acid; and 3) the bulkiness of the side
chain or any one or more of these characteristics.
Illustratively, the well-known terminologies "hydrophilic residues"
relate to serine or threonine. "Hydrophobic residues" refer to
leucine, isoleucine, phenylalanine, valine or alanine, or the like.
"Positively charged residues" relate to lysine, arginine,
ornithine, or histidine. "Negatively charged residues" refer to
aspartic acid or glutamic acid. Residues having "bulky side chains"
refer to phenylalanine, tryptophan or tyrosine, or the like. A list
of illustrative conservative amino acid substitutions is given in
TABLE 1.
TABLE-US-00016 TABLE 1 For Amino Acid Replace With Alanine D-Ala,
Gly, Aib, .beta.-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn
D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D- Gln Aspartic
Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D- Gln Cysteine D-Cys,
S--Me-Cys, Met, D-Met, Thr, D- Thr Glutamine D-Gln, Asn, D-Asn,
Glu, D-Glu, Asp, D- Asp Glutamic Acid D-Glu, D-Asp, Asp, Asn,
D-Asn, Gln, D- Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, .beta.-Ala
Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D- Met Leucine Val,
D-Val, Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn,
D-Orn Methionine D-Met, S--Me-Cys, Ile, D-Ile, Leu, D-Leu, Val,
D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D- Trp
Proline D-Pro Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys
Threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val
Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D- Trp Valine D-Val,
Leu, D-Leu, Ile, D-Ile, Met, D- Met
In one embodiment, the conservative amino acid substitutions
appicable to the molecules described herein do not alter the motifs
that consist of the B1-X1-X2-X3-X4-X5-X6-X7-X8-B2 formula, the
B1-X1-B2-X2-X3--X4-X5-X6-X7-X8-X9-B3 formula, the
B1-X1-X2-X3-X4-X5-X6-X7-X8-B2-X9-B3 formula, or the Arg-Arg
motif.
[0305] In various embodiments described herein, the glycan (e.g.
glycosaminoglycan, abbreviated GAG, or polysaccharide) component of
the synthetic peptidoglycan described herein can be selected from
the group consisting of dextran, chondroitin, chondroitin sulfate,
dermatan, dermatan sulfate, heparan, heparin, keratin, keratan
sulfate, and hyaluronic acid. In one embodiment, the glycan is
selected from the group consisting of chondroitin sulfate and
keratan sulfate. In another illustrative embodiment, the glycan is
chondroitin sulfate.
[0306] In one embodiment described herein, the hyaluronic
acid-binding synthetic peptidoglycan comprises
(GAHWQFNALTVRGG).sub.10 conjugated to chondroitin sulfate wherein
each peptide in the peptidoglycan molecule is linked separately to
chondroitin sulfate. In another embodiment described herein, the
hyaluronic acid-binding synthetic peptidoglycan comprises
(GAHWQFNALTVRGGGC).sub.11 conjugated to chondroitin sulfate wherein
each peptide in the peptidoglycan molecule is linked separately to
chondroitin sulfate. In each of the above peptide embodiments, the
peptide number may be selected from the group consisting of 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, and 25 peptide molecules.
[0307] In various embodiments described herein, the synthetic
peptidoglycan is resistant to aggrecanase. An aggrecanase is
characterized in the art as any enzyme known to cleave
aggrecan.
[0308] In one illustrative aspect, the hyaluronic acid-binding
synthetic peptidoglycan may be sterilized. As used herein
"sterilization" or "sterilize" or "sterilized" means disinfecting
the hyaluronic acid-binding synthetic peptidoglycans by removing
unwanted contaminants including, but not limited to, endotoxins and
infectious agents.
[0309] In various illustrative embodiments, the hyaluronic
acid-binding synthetic peptidoglycan can be disinfected and/or
sterilized using conventional sterilization techniques including
propylene oxide or ethylene oxide treatment, gas plasma
sterilization, gamma radiation (e.g., 1-4 Mrads gamma irradiation
or 1-2.5 Mrads of gamma irradiation), electron beam, and/or
sterilization with a peracid, such as peracetic acid. Sterilization
techniques which do not adversely affect the structure and
biotropic properties of the hyaluronic acid-binding synthetic
peptidoglycan can be used. In one embodiment, the hyaluronic
acid-binding synthetic peptidoglycan can be subjected to one or
more sterilization processes. In another illustrative embodiment,
the hyaluronic acid-binding synthetic peptidoglycan is subjected to
sterile filtration. The hyaluronic acid-binding synthetic
peptidoglycan may be wrapped in any type of container including a
plastic wrap or a foil wrap, and may be further sterilized. The
hyaluronic acid-binding synthetic peptidoglycan may be prepared
under sterile conditions, for example, by lyophilisation, which may
readily be accomplished using standard techniques well-known to
those skilled in the art.
[0310] In various embodiments described herein, the hyaluronic
acid-binding synthetic peptidoglycans can be combined with
minerals, amino acids, sugars, peptides, proteins, vitamins (such
as ascorbic acid), or laminin, collagen, fibronectin, hyaluronic
acid, fibrin, elastin, or aggrecan, or growth factors such as
epidermal growth factor, platelet-derived growth factor,
transforming growth factor beta, or fibroblast growth factor, and
glucocorticoids such as dexamethasone or viscoelastic altering
agents, such as ionic and non-ionic water soluble polymers; acrylic
acid polymers; hydrophilic polymers such as polyethylene oxides,
polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol;
cellulosic polymers and cellulosic polymer derivatives such as
hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl
methylcellulose, hydroxypropyl methylcellulose phthalate, methyl
cellulose, carboxymethyl cellulose, and etherified cellulose;
poly(lactic acid), poly(glycolic acid), copolymers of lactic and
glycolic acids, or other polymeric agents both natural and
synthetic.
[0311] In various embodiments described herein, the peptide
component of the synthetic peptidoglycan is synthesized according
to solid phase peptide synthesis protocols that are well-known by
persons of skill in the art. In one embodiment a peptide precursor
is synthesized on a solid support according to the well-known Fmoc
protocol, cleaved from the support with trifluoroacetic acid and
purified by chromatography according to methods known to persons
skilled in the art.
[0312] In various embodiments described herein, the peptide
component of the synthetic peptidoglycan is synthesized utilizing
the methods of biotechnology that are well-known to persons skilled
in the art. In one embodiment a DNA sequence that encodes the amino
acid sequence information for the desired peptide is ligated by
recombinant DNA techniques known to persons skilled in the art into
an expression plasmid (for example, a plasmid that incorporates an
affinity tag for affinity purification of the peptide), the plasmid
is transfected into a host organism for expression, and the peptide
is then isolated from the host organism or the growth medium
according to methods known by persons skilled in the art (e.g., by
affinity purification). Recombinant DNA technology methods are
described in Sambrook et al., "Molecular Cloning: A Laboratory
Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001),
incorporated herein by reference, and are well-known to the skilled
artisan.
[0313] In various embodiments described herein, the peptide
component of the hyaluronic acid-binding synthetic peptidoglycan is
conjugated to a glycan by reacting a free amino group of the
peptide with an aldehyde function of the glycan in the presence of
a reducing agent, utilizing methods known to persons skilled in the
art, to yield the peptide glycan conjugate. In one embodiment an
aldehyde function of the glycan (e.g. polysaccharide or
glycosaminoglycan) is formed by reacting the glycan with sodium
metaperiodate according to methods known to persons skilled in the
art.
[0314] In one embodiment, the peptide component of the synthetic
peptidoglycan is conjugated to a glycan by reacting an aldehyde
function of the glycan with 3-(2-pyridyldithio)propionyl hydrazide
(PDPH) to form an intermediate glycan and further reacting the
intermediate glycan with a peptide containing a free thiol group to
yield the peptide glycan conjugate. In yet another embodiment, the
sequence of the peptide component of the synthetic peptidoglycan
may be modified to include a glycine-cysteine segment to provide an
attachment point for a glycan or a glycan-linker conjugate. In any
of the embodiments described herein, the crosslinker can be
N[.beta.-Maleimidopropionic acid]hydrazide (BMPH).
[0315] Although specific embodiments have been described in the
preceding paragraphs, the hyaluronic acid-binding synthetic
peptidoglycans described herein can be made by using any
art-recognized method for conjugation of the peptide to the glycan
(e.g. polysaccharide or glycosaminoglycan). This can include
covalent, ionic, or hydrogen bonding, either directly or indirectly
via a linking group such as a divalent linker. The conjugate is
typically formed by covalent bonding of the peptide to the glycan
through the formation of amide, ester or imino bonds between acid,
aldehyde, hydroxy, amino, or hydrazo groups on the respective
components of the conjugate. All of these methods are known in the
art or are further described in the Examples section of this
application or in Hermanson G. T., Bioconjugate Techniques,
Academic Press, pp. 169-186 (1996), incorporated herein by
reference. The linker typically comprises about 1 to about 30
carbon atoms, more typically about 2 to about 20 carbon atoms.
Lower molecular weight linkers (i.e., those having an approximate
molecular weight of about 20 to about 500) are typically
employed.
[0316] In addition, structural modifications of the linker portion
of the conjugates are contemplated herein. For example, amino acids
may be included in the linker and a number of amino acid
substitutions may be made to the linker portion of the conjugate,
including but not limited to naturally occurring amino acids, as
well as those available from conventional synthetic methods. In
another aspect, beta, gamma, and longer chain amino acids may be
used in place of one or more alpha amino acids. In another aspect,
the linker may be shortened or lengthened, either by changing the
number of amino acids included therein, or by including more or
fewer beta, gamma, or longer chain amino acids. Similarly, the
length and shape of other chemical fragments of the linkers
described herein may be modified.
[0317] In various embodiments described herein, the linker may
include one or more bivalent fragments selected independently in
each instance from the group consisting of alkylene,
heteroalkylenc, cycloalkylene, cycloheteroalkylene, arylenc, and
heteroarylenc each of which is optionally substituted. As used
herein heteroalkylene represents a group resulting from the
replacement of one or more carbon atoms in a linear or branched
alkylene group with an atom independently selected in each instance
from the group consisting of oxygen, nitrogen, phosphorus and
sulfur. In an alternative embodiment, a linker is not present.
[0318] In one embodiment described herein, an engineered collagen
matrix is provided. The previously described embodiments of the
hyaluronic acid-binding synthetic peptidoglycan are applicable to
the engineered collagen matrix described herein. In one embodiment,
the engineered collagen matrix comprises polymerized collagen,
hyaluronic acid, and a hyaluronic acid-binding synthetic
peptidoglycan. In one embodiment, the engineered collagen matrix
comprises polymerized collagen and a hyaluronic-binding synthetic
peptidoglycan. In various illustrative embodiments, crosslinking
agents, such as carbodiimides, aldehydes, lysl-oxidase,
N-hydroxysuccinimide esters, imidoesters, hydrazides, and
maleimides, as well as various natural crosslinking agents,
including genipin, and the like, can be added before, during, or
after polymerization of the collagen in solution.
[0319] In various illustrative embodiments, the collagen used
herein to prepare an engineered collagen matrix may be any type of
collagen, including collagen types I to XXVIII, alone or in any
combination, for example, collagen types I, II, III, and/or IV may
be used. In some embodiments, the collagen used to prepare an
engineered collagen matrix is selected from the group consisting of
type I collagen, type II collagen, type III collagen, type IV
collagen, type IX collagen, type XI collagen, and combinations
thereof. In one embodiment, the engineered collagen matrix is
formed using commercially available collagen (e.g., Sigma, St.
Louis, Mo.). In an alternative embodiment, the collagen can be
purified from submucosa-containing tissue material such as
intestinal, urinary bladder, or stomach tissue. In a further
embodiment, the collagen can be purified from tail tendon. In an
additional embodiment, the collagen can be purified from skin. In
various aspects, the collagen can also contain endogenous or
exogenously added non-collagenous proteins in addition to the
collagen-binding synthetic peptidoglycans, such as fibronectin or
silk proteins, glycoproteins, and polysaccharides, or the like. The
engineered collagen matrices prepared by the methods described
herein can be in the form of a tissue graft (e.g., in the form of a
gel) which can assume the characterizing features of the tissue(s)
with which they are associated at the site of implantation or
injection. In one embodiment, the engineered collagen matrix is a
tissue graft that can be implanted into a patient. In another
embodiment, the engineered collagen matrix can be administered to a
patient by injection. In either embodiment, the matrix can be in
the form of a gel or a powder, for example.
[0320] In one embodiment, the collagen in the engineered collagen
matrix comprises about 40 to about 90 dry weight (wt) % of the
matrix, about 40 to about 80 dry wt % of the matrix, about 40 to
about 70 dry wt % of the matrix, about 40 to about 60 dry wt % of
the matrix, about 50 to about 90 dry wt % of the matrix, about 50
to about 80 dry wt % of the matrix, about 50 to about 75 dry wt %
of the matrix, about 50 to about 70 dry wt % of the matrix, or
about 60 to about 75 dry wt % of the matrix. In another embodiment,
the collagen in the engineered collagen matrix comprises about 90
dry wt %, about 85 dry wt %, about 80 dry wt %, about 75 dry wt %,
about 70 dry wt %, about 65 dry wt %, about 60 dry wt %, about 50
dry wt %, about 45 dry wt %, about 40 dry wt %, or about 30 dry wt
% of the matrix.
[0321] In one embodiment, the final collagen concentration of the
matrix in gel form is about 0.5 to about 6 mg per mL, about 0.5 to
about 5 mg per mL, about 0.5 to about 4 mg per mL, about 1 to about
6 mg per mL, about 1 to about 5 mg per mL, or about 1 to about 4 mg
per mL. In one embodiment, the final collagen concentration of the
matrix is about 0.5 mg per mL, about 1 mg per mL, about 2 mg per
mL, about 3 mg per mL, about 4 mg per mL, or about 5 mg per mL.
[0322] In one embodiment, the hyaluronic acid-binding synthetic
peptidoglycan in the engineered collagen matrix comprises about 2
to about 60 dry weight (wt) % of the matrix, about 2 to about 50
dry wt % of the matrix, about 5 to about 50 dry wt % of the matrix,
about 10 to about 50 dry wt % of the matrix, about 10 to about 20
dry wt % of the matrix, about 10 to about 30 dry wt % of the
matrix, about 10 to about 25 dry wt % of the matrix, about 15 to
about 30 dry wt % of the matrix, or about 15 to about 45 dry wt %
of the matrix. In another embodiment, the hyaluronic acid-binding
synthetic peptidoglycan in the engineered collagen matrix comprises
about 2 dry wt %, about 5 dry wt %, about 10 dry wt %, about 15 dry
wt %, about 20 dry wt %, about 25 dry wt %, about 30 dry wt %,
about 35 dry wt %, about 40 dry wt %, about 45 dry wt %, or about
50 dry wt % of the matrix
[0323] In another embodiment, the engineered collagen matrix
comprises hyaluronic acid and the hyaluronic acid in the engineered
collagen matrix comprises about 2 to about 60 dry weight (wt) % of
the matrix, about 2 to about 50 dry wt % of the matrix, about 5 to
about 50 dry wt % of the matrix, about 10 to about 50 dry wt % of
the matrix, about 10 to about 20 dry wt % of the matrix, about 10
to about 30 dry wt % of the matrix, about 10 to about 25 dry wt %
of the matrix, about 15 to about 30 dry wt % of the matrix, or
about 15 to about 45 dry wt % of the matrix. In another embodiment,
the hyaluronic acid in the engineered collagen matrix comprises
about 2 dry wt %, about 5 dry wt %, about 10 dry wt %, about 15 dry
wt %, about 20 dry wt %, about 25 dry wt %, about 30 dry wt %,
about 35 dry wt %, about 40 dry wt %, about 45 dry wt %, or about
50 dry wt % of the matrix.
[0324] In one embodiment, the engineered collagen matrix comprises
hyaluronic acid and a hyaluronic acid-binding synthetic
peptidoglycan. The hyaluronic acid and hyaluronic acid-binding
synthetic peptidoglycan in the engineered collagen matrix comprise
about 10 to about 60 dry weight (wt) % of the matrix, about 20 to
about 60 dry wt % of the matrix, about 30 to about 60 dry wt % of
the matrix, about 40 to about 60 dry wt % of the matrix, about 10
to about 50 dry wt % of the matrix, about 20 to about 50 dry wt %
of the matrix, about 25 to about 50 dry wt % of the matrix, about
30 to about 50 dry wt % of the matrix, or about 25 to about 40 dry
wt % of the matrix. In another embodiment, the hyaluronic acid and
hyaluronic acid-binding synthetic peptidoglycan in the engineered
collagen matrix comprises about 10 dry wt %, about 15 dry wt %,
about 20 dry wt %, about 25 dry wt %, about 30 dry wt %, about 35
dry wt %, about 40 dry wt %, about 50 dry wt %, about 55 dry wt %,
about 60 dry wt %, or about 70 dry wt % of the matrix.
[0325] In one illustrative aspect, the engineered collagen matrix
may be sterilized. As used herein "sterilization" or "sterilize" or
"sterilized" means disinfecting the matrix by removing unwanted
contaminants including, but not limited to, endotoxins, nucleic
acid contaminants, and infectious agents.
[0326] In various illustrative embodiments, the engineered collagen
matrix can be disinfected and/or sterilized using conventional
sterilization techniques including glutaraldehyde tanning,
formaldehyde tanning at acidic pH, propylene oxide or ethylene
oxide treatment, gas plasma sterilization, gamma radiation (e.g.,
1-4 Mrads gamma irradiation or 1-2.5 Mrads of gamma irradiation),
electron beam, and/or sterilization with a peracid, such as
peracetic acid. Sterilization techniques which do not adversely
affect the structure and biotropic properties of the matrix can be
used. In one embodiment, the engineered collagen matrix can be
subjected to one or more sterilization processes. In illustrative
embodiments, the collagen in solution, prior to polymerization, can
also be sterilized or disinfected. The engineered collagen matrix
may be wrapped in any type of container including a plastic wrap or
a foil wrap, and may be further sterilized.
[0327] In any of these embodiments the engineered collagen matrix
may further comprise an exogenous population of cells. The added
population of cells may comprise one or more cell populations. In
various embodiments, the cell populations comprise a population of
non-keratinized or keratinized epithelial cells or a population of
cells selected from the group consisting of endothelial cells,
mesodermally derived cells, mesothelial cells, synoviocytes, neural
cells, glial cells, osteoblasts, fibroblasts, chondrocytes,
tenocytes, smooth muscle cells, skeletal muscle cells, cardiac
muscle cells, multi-potential progenitor cells (e.g., stem cells,
including bone marrow progenitor cells), and osteogenic cells. In
some embodiments, the population of cells is selected from the
group consisting of chondrocytes and stem cells. In some
embodiments, the stem cells are selected from the group consisting
of osteoblasts, osteogenic cells, and mesenchymal stem cells. In
various embodiments, the engineered collagen matrix can be seeded
with one or more cell types in combination.
[0328] In various aspects, the engineered collagen matrices or
engineered graft constructs of the present invention can be
combined with nutrients, including minerals, amino acids, sugars,
peptides, proteins, vitamins (such as ascorbic acid), or laminin,
fibronectin, hyaluronic acid, fibrin, elastin, or aggrecan, or
growth factors such as epidermal growth factor, platelet-derived
growth factor, transforming growth factor beta, or fibroblast
growth factor, and glucocorticoids such as dexamethasone or
viscoelastic altering agents, such as ionic and non-ionic water
soluble polymers; acrylic acid polymers; hydrophilic polymers such
as polyethylene oxides, polyoxyethylene-polyoxypropylene
copolymers, and polyvinylalcohol; cellulosic polymers and
cellulosic polymer derivatives such as hydroxypropyl cellulose,
hydroxyethyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate, methyl cellulose,
carboxymethyl cellulose, and etherified cellulose; poly(lactic
acid), poly(glycolic acid), copolymers of lactic and glycolic
acids, or other polymeric agents both natural and synthetic. In
other illustrative embodiments, cross-linking agents, such as
carbodiimides, aldehydes, lysl-oxidase, N-hydroxysuccinimide
esters, imidoesters, hydrazides, and maleimides, as well as natural
crosslinking agents, including genipin, and the like can be added
before, concurrent with, or after the addition of cells.
[0329] As discussed above, in accordance with one embodiment, cells
can be added to the engineered collagen matrices or the engineered
graft constructs after polymerization of the collagen or during
collagen polymerization. The engineered collagen matrices
comprising the cells can be subsequently injected or implanted in a
host for use as engineered graft constructs. In another embodiment,
the cells on or within the engineered collagen matrices can be
cultured in vitro, for a predetermined length of time, to increase
the cell number or to induce desired remodeling prior to
implantation or injection into a patient.
[0330] In one embodiment described herein, a composition for in
vitro culture of chondrocytes or stem cells is provided (i.e., for
in vitro culture of cells without subsequent implantation or
injection into a patient). The composition for in vitro culture
comprises a hyaluronic acid-binding synthetic peptidoglycan. The
previously described embodiments of the hyaluronic acid-binding
synthetic peptidoglycan are applicable to the composition for in
vitro culture described herein.
[0331] In various aspects, the composition for in vitro culture of
the present invention can be combined with nutrients, including
minerals, amino acids, sugars, peptides, proteins, vitamins (such
as ascorbic acid), or laminin, fibronectin, hyaluronic acid,
fibrin, elastin, or aggrecan, or growth factors such as epidermal
growth factor, platelet-derived growth factor, transforming growth
factor beta, or fibroblast growth factor, and glucocorticoids such
as dexamethasone.
[0332] In some embodiments, the composition for in vitro culture
includes stem cells selected from the group consisting of
osteoblasts, osteogenic cells, and mesenchymal stem cells. In
various embodiments, the composition for in vitro culture can be
seeded with one or more cell types in combination.
[0333] In one illustrative aspect, the composition for in vitro
culture may be sterilized. As used herein "sterilization" or
"sterilize" or "sterilized" means disinfecting the composition by
removing unwanted contaminants including, but not limited to,
endotoxins, nucleic acid contaminants, and infectious agents. The
sterilization procedures, methods and embodiments provided in the
preceding paragraphs are also applicable to the composition for in
vitro culture described herein. The in vitro culture composition
may be used to expand populations of cells for implantation or
injection into a patient.
[0334] In one embodiment described herein, an additive for a
biomaterial cartilage replacement composition is provided. The
additive comprises a hyaluronic acid-binding synthetic
peptidoglycan for addition to an existing biomaterial cartilage
replacement material. The previously described embodiments of the
hyaluronic acid-binding synthetic peptidoglycan are applicable to
the additive described herein.
[0335] As used herein, the phrase "existing biomaterial cartilage
replacement material" means a biologically compatible composition
that can be utilized for replacement of damaged, defective, or
missing cartilage in the body. Various types of existing
biomaterial cartilage replacement compositions are well-known in
the art and are contemplated. For example, existing biomaterial
cartilage or bone replacement compositions include the DeNovo.RTM.
NT Natural Tissue Graft (Zimmer), MaioRegen.TM. (JRI Limited), or
the collection of cryopreserved osteoarticular tissues produced by
Biomet.
[0336] In one embodiment, a method of preparing a biomaterial or
bone cartilage replacement is provided. The method comprises the
step of combining the synthetic peptidoglycan and an existing
biomaterial or bone cartilage replacement material. The previously
described embodiments of the hyaluronic acid-binding synthetic
peptidoglycan are applicable to the method described herein.
[0337] In one embodiment, a method of treatment for arthritis in a
patient is provided. The method comprises the step of administering
to the patient a hyaluronic acid-binding synthetic peptidoglycan,
wherein the synthetic peptidoglycan reduces one or more symptoms
associated with arthritis. The previously described embodiments of
the hyaluronic acid-binding synthetic peptidoglycan are applicable
to the method described herein.
[0338] In various embodiments, the synthetic peptidoglycan used in
the method of treatment for arthritis reduces one or more symptoms
associated with arthritis. Various symptoms are known in the art to
be associated with arthritis, including but not limited to pain,
stiffness, tenderness, inflammation, swelling, redness, warmth, and
decreased mobility. The symptoms of arthritis may be present in a
joint, a tendon, or other parts of the body. As used herein,
"reducing" means preventing or completely or partially alleviating
a symptom of arthritis.
[0339] In various embodiments, the arthritis is osteoarthritis or
rheumatoid arthritis. The pathogenesis and clinical symptoms of
osteoarthritis and rheumatoid arthritis are well-known in the art.
In one embodiment of this method, the synthetic peptidoglycan acts
as a lubricant following administration or prevents loss of
cartilage. In another embodiment, the synthetic peptidoglycan
prevents articulation of bones in the patient. For example, the
synthetic peptidoglycan inhibits bone on bone articulation in a
patient with reduced or damaged cartilage.
[0340] In one embodiment, a method of reducing or preventing
degradation of ECM components in a patient is provided. For
example, a method of reducing or preventing degradation of ECM
components in the cartilage of a patient is provided. The method
comprises administering to the patient a hyaluronic acid-binding
synthetic peptidoglycan. The previously described embodiments of
the hyaluronic acid-binding synthetic peptidoglycan are applicable
to the method described herein. In one embodiment, the synthetic
peptidoglycan is resistant to matrix metallo proteases, e.g., an
aggrecanase.
[0341] In another embodiment, a method of reducing or preventing
hyaluronic acid degradation in a patient is provided. The method
comprises administering to the patient a hyaluronic acid-binding
synthetic peptidoglycan. The previously described embodiments of
the hyaluronic acid-binding synthetic peptidoglycan are applicable
to the method described herein.
[0342] In another embodiment, a method of reducing or preventing
collagen degradation is provided. The method comprises the steps of
contacting a hyaluronic acid-binding synthetic peptidoglycan with
hyaluronic acid in the presence of collagen, and reducing or
preventing collagen degradation. The previously described
embodiments of the hyaluronic acid-binding synthetic peptidoglycan
arc applicable to the method described herein.
[0343] In another embodiment, a method of reducing or preventing
chondroitin sulfate degradation is provided. The method comprises
the steps of contacting a hyaluronic acid-binding synthetic
peptidoglycan with hyaluronic acid in the presence of collagen, and
reducing or preventing chondroitin sulfate degradation. The
previously described embodiments of the hyaluronic acid-binding
synthetic peptidoglycan are applicable to the method described
herein.
[0344] "Reducing" ECM component degradation, e.g., hyaluronic acid,
collagen, or chondroitin sulfate degradation, means completely or
partially reducing degradation of hyaluronic acid, collagen, or
chondroitin sulfate, respectively.
[0345] In one embodiment, reducing hyaluronic acid degradation in a
patient means reducing the rate of hyaluronic acid degradation. For
example, FIG. 8 described in the Examples section of the
application shows that the rate of hyaluronic acid degradation in a
mixture of hyaluronic acid and a hyaluronic acid-binding synthetic
peptidoglycan is significantly reduced upon addition of the
synthetic peptidoglycan.
[0346] In one embodiment, reducing collagen degradation means
reducing the rate of collagen degradation. For example, FIG. 10
described in the Examples section of the application shows that the
rate of collagen degradation in the presence of hyaluronic acid and
a hyaluronic acid-binding synthetic peptidoglycan is significantly
reduced upon addition of the synthetic peptidoglycan.
[0347] In one embodiment, reducing chondroitin sulfate degradation
means reducing the rate of chondroitin sulfate degradation. For
example, FIG. 11 described in the Examples section of the
application shows that the rate of chondroitin sulfate degradation
in the presence of a hyaluronic acid-binding synthetic
peptidoglycan is significantly reduced upon addition of the
synthetic peptidoglycan.
[0348] In one embodiment described herein, a method for correcting
or modifying a tissue defect in a patient is provided. The method
comprises administering into the tissue defect hyaluronic acid and
a hyaluronic acid-binding synthetic peptidoglycan wherein the
defect is corrected or modified. The previously described
embodiments of the hyaluronic acid-binding synthetic peptidoglycan
are applicable to the method described herein. In one embodiment,
the tissue defect is a cosmetic defect.
[0349] The following embodiments are applicable to methods
described herein where the hyaluronic acid-binding synthetic
peptidoglycan is administered to a patient. In various embodiments,
the hyaluronic acid-binding synthetic peptidoglycan can be injected
or implanted (e.g., incorporated in a cartilage repair composition
or device). In some embodiments described herein, the injection is
an intraarticular injection. In another embodiment described
herein, the injection is into a joint capsule of the patient. In
other embodiments, the injection is a subcutaneous injection, as in
the case of dermal fillers. Suitable means for injection include a
needle (including microneedle) injector or a device for
infusion.
[0350] In an illustrative embodiment, pharmaceutical formulations
for use with hyaluronic acid-binding synthetic peptidoglycans for
administration to a patient comprise: a) a pharmaceutically active
amount of the hyaluronic acid-binding synthetic peptidoglycan; b) a
pharmaceutically acceptable pH buffering agent to provide a pH in
the range of about pH 4.5 to about pH 9; c) an ionic strength
modifying agent in the concentration range of about 0 to about 300
millimolar; and d) water soluble viscosity modifying agent in the
concentration range of about 0.25% to about 10% total formula
weight or any individual component a), b), c), or d) or any
combinations of a), b), c) and d).
[0351] In various embodiments described herein, the pH buffering
agents are those agents known to the skilled artisan and include,
for example, acetate, borate, carbonate, citrate, and phosphate
buffers, as well as hydrochloric acid, sodium hydroxide, magnesium
oxide, monopotassium phosphate, bicarbonate, ammonia, carbonic
acid, hydrochloric acid, sodium citrate, citric acid, acetic acid,
disodium hydrogen phosphate, borax, boric acid, sodium hydroxide,
diethyl barbituric acid, and proteins, as well as various
biological buffers, for example, TAPS, Bicine, Tris, Tricine,
HEPES, TES, MOPS, PIPES, cacodylate, or MES.
[0352] In various embodiments described herein, the ionic strength
modifying agents include those agents known in the art, for
example, glycerin, propylene glycol, mannitol, glucose, dextrose,
sorbitol, sodium chloride, potassium chloride, and other
electrolytes.
[0353] Useful viscosity modulating agents include but are not
limited to, ionic and non-ionic water soluble polymers; crosslinked
acrylic acid polymers such as the "carbomer" family of polymers,
e.g., carboxypolyalkylenes that may be obtained commercially under
the Carbopol.RTM. trademark; hydrophilic polymers such as
polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers,
and polyvinylalcohol; cellulosic polymers and cellulosic polymer
derivatives such as hydroxypropyl cellulose, hydroxyethyl
cellulose, hydroxypropyl methylcellulose, hydroxypropyl
methylcellulose phthalate, methyl cellulose, carboxymethyl
cellulose, and etherified cellulose; gums such as tragacanth and
xanthan gum; sodium alginate; gelatin, hyaluronic acid and salts
thereof, chitosans, gellans or any combination thereof. Typically,
non-acidic viscosity enhancing agents, such as a neutral or basic
agent are employed in order to facilitate achieving the desired pH
of the formulation.
[0354] In various embodiments described herein, formulations for
injection may be suitably formulated as a sterile non-aqueous
solution or as a dried form (e.g., lyophilized) to be used in
conjunction with a suitable vehicle such as sterile, pyrogen-free
water. The preparation of formulations for injection under sterile
conditions, for example, by lyophilisation, may readily be
accomplished using standard pharmaceutical techniques well-known to
those skilled in the art. In one embodiment, the viscosity of a
solution containing hyaluronic acid is increased by addition of a
hyaluronic acid-binding synthetic peptidoglycan.
[0355] In various embodiments described herein, the solubility of a
hyaluronic acid-binding synthetic peptidoglycan used in the
preparation of formulations for administration via injection may be
increased by the use of appropriate formulation techniques, such as
the incorporation of solubility-enhancing compositions such as
mannitol, ethanol, glycerin, polyethylene glycols, propylene
glycol, poloxomers, and others known to those of skill in the
art.
[0356] In various embodiments described herein, formulations for
administration via injection may be formulated to be for immediate
and/or modified release. Modified release formulations include
delayed, sustained, pulsed, controlled, targeted and programmed
release formulations. Thus, a hyaluronic acid-binding synthetic
peptidoglycan may be formulated as a solid, semi-solid, or
thixotropic liquid for administration as an implanted depot
providing modified release of the active compound. Illustrative
examples of such formulations include drug-coated stents and
copolymeric(dl-lactic, glycolic)acid (PGLA) microspheres. In
another embodiment, hyaluronic acid-binding synthetic
peptidoglycans or compositions comprising hyaluronic acid-binding
synthetic peptidoglycan may be continuously administered, where
appropriate.
[0357] In any of the embodiments described herein, the hyaluronic
acid-binding synthetic peptidoglycan can be administered alone or
in combination with suitable pharmaceutical carriers or diluents.
Diluent or carrier ingredients used in the hyaluronic acid-binding
synthetic peptidoglycan formulation can be selected so that they do
not diminish the desired effects of the hyaluronic acid-binding
synthetic peptidoglycan. The hyaluronic acid- binding synthetic
peptidoglycan formulation may be in any suitable form. Examples of
suitable dosage forms include aqueous solutions of the hyaluronic
acid-binding synthetic peptidoglycan, for example, a solution in
isotonic saline, 5% glucose or other well-known pharmaceutically
acceptable liquid carriers such as alcohols, glycols, esters and
amides.
[0358] Suitable dosages of the hyaluronic acid-binding synthetic
peptidoglycan can be determined by standard methods, for example by
establishing dose-response curves in laboratory animal models or in
clinical trials. In various embodiments described herein, the
dosage of the hyaluronic acid-binding synthetic peptidoglycan, can
vary significantly depending on the patient condition, the disease
state being treated, the route of administration and tissue
distribution, and the possibility of co-usage of other therapeutic
treatments. Illustratively, suitable dosages of hyaluronic
acid-binding synthetic peptidoglycan (administered in a single
bolus or over time) include from about 1 ng/kg to about 10 mg/kg,
from about 100 ng/kg to about 1 mg/kg, from about 1 .mu.g/kg to
about 500 .mu.g/kg, or from about 100 .mu.g/kg to about 400
.mu.g/kg. In each of these embodiments, dose/kg refers to the dose
per kilogram of patient mass or body weight. In other illustrative
aspects, effective doses can range from about 0.01 .mu.g to about
1000 mg per dose, from about 1 .mu.g to about 100 mg per dose, or
from about 100 .mu.g to about 50 mg per dose, or from about 500
.mu.g to about 10 mg per dose, or from about 1 mg to 10 mg per
dose, or from about 1 to about 100 mg per dose, or from about 1 mg
to 5000 mg per dose, or from about 1 mg to 3000 mg per dose, or
from about 100 mg to 3000 mg per dose, or from about 1000 mg to
3000 mg per dose. In one embodiment, suitable dosages of a
hyaluronic acid-binding synthetic peptidoglycan include
concentrations ranging from about 0.01 uM to about 100 uM, about
0.05 to about 100 uM, about 0.1 uM to about 100 uM, about 0.1 uM to
about 50 uM, about 0.1 uM to about 20 uM, about 0.1 uM to about 10
uM, about 0.5 uM to about 10 uM, about 0.5 uM to about 50 uM, and
about 0.5 uM to about 100 uM. In another embodiment, suitable
dosages of a hyaluronic acid-binding synthetic peptidoglycan
include concentrations of about 0.01 uM, 0.1 uM, 0.2 uM, 0.5 uM, 1
uM, 2 uM, 5 uM, 10 uM, 20 uM, 50 uM, and 100 uM.
[0359] The hyaluronic acid-binding synthetic peptidoglycan can be
formulated in an excipient. In any of the embodiments described
herein, the excipient can have a concentration ranging from about
0.4 mg/ml to about 6 mg/ml. In various embodiments, the
concentration of the excipient may range from about 0.5 mg/ml to
about 10 mg/ml, from about 0.1 mg/ml to about 6 mg/ml, from about
0.5 mg/ml to about 3 mg/ml, from about 1 mg/ml to about 3 mg/ml,
from about 0.01 mg/ml to about 10 mg/ml, and from about 2 mg/ml to
about 4 mg/ml.
[0360] In embodiments where the hyaluronic acid-binding synthetic
peptidoglycan is implanted as part of a cartilage repair
composition or device (e.g., a gel for implantation), any suitable
formulation described above may be used.
[0361] Any effective regimen for administering the hyaluronic
acid-binding synthetic peptidoglycan can be used. For example, the
hyaluronic acid-binding synthetic peptidoglycan can be administered
as a single dose, or as a multiple-dose daily regimen. Further, a
staggered regimen, for example, one to five days per week can be
used as an alternative to daily treatment.
[0362] In various embodiments described herein, the patient is
treated with multiple injections of the hyaluronic acid-binding
synthetic peptidoglycan. In one embodiment, the patient is injected
multiple times (e.g., about 2 up to about 50 times) with the
hyaluronic acid- binding synthetic peptidoglycan, for example, at
12-72 hour intervals or at 48-72 hour intervals. Additional
injections of the hyaluronic acid-binding synthetic peptidoglycan
can be administered to the patient at an interval of days or months
after the initial injections(s).
[0363] In any of the embodiments herein described, it is to be
understood that a combination of two or more hyaluronic
acid-binding synthetic peptidoglycans, differing in the peptide
portion, the glycan portion, or both, can be used in place of a
single hyaluronic acid-binding synthetic peptidoglycan.
[0364] It is also appreciated that in the foregoing embodiments,
certain aspects of the compounds, compositions and methods are
presented in the alternative in lists, such as, illustratively,
selections for any one or more of G and P. It is therefore to be
understood that various alternate embodiments of the invention
include individual members of those lists, as well as the various
subsets of those lists. Each of those combinations is to be
understood to be described herein by way of the lists.
[0365] In the following illustrative examples, the terms "aggrecan
mimetic" and "mimetic" are used synonymously with the term
"hyaluronic acid-binding synthetic peptidoglycan."
EXAMPLE 1
Peptide Synthesis
[0366] All peptides were synthesized using a Symphony peptide
synthesizer (Protein Technologies, Tucson, Ariz.), utilizing an
FMOC protocol on a Knorr resin. The crude peptide was released from
the resin with TFA and purified by reverse phase chromatography on
an AKTAexplorer (GE Healthcare, Piscataway, N.J.) utilizing a
Grace-Vydac 218TP C-18 reverse phase column and a gradient of
water/acetonitrile 0.1%TFA. Dansyl-modified peptides were prepared
by adding an additional coupling step with dansyl-Gly (Sigma)
before release from the resin. Peptide structures were confirmed by
mass spectrometry. The following peptides were prepared as
described above: GAHWQFNALTVRGGGC, KQKIKHVVKLKGC, and
KLKSQLVKRKGC.
EXAMPLE 2
Chondroitin Sulfate Functionalization and Synthetic Peptidoglycan
Formation
[0367] The reaction schematic for the creation of the aggrecan
mimic (i.e., GAH) can be seen in FIG. 1. Functionalization of the
chondroitin sulfate (CS) (Sigma, St. Louis, Mo.) was accomplished
using sodium periodate (Thermo Scientific, Waltham, Mass.) to
oxidize the CS. By varying the reaction duration and sodium
periodate concentration, the number of aldehyde groups produced by
the oxidation reaction was controlled, values presented in Table 2.
Table 2 details the sodium periodate concentration and the reaction
duration needed to obtain the desired number of aldehydes per CS
chain. Through progressive chemical reactions, schematic shown in
FIG. 1, the number of BMPH attached per CS chain is assumed to
equal the number of aldehydes produced and the number of hyaluronic
acid (HA) binding peptides attached.
Based on the reaction duration and the concentration of sodium
periodate, the number of peptides (average) per CS chain is shown
in Table 2.
TABLE-US-00017 TABLE 2 Sodium Periodate Reaction Concentration
Duration # Aldehydes/ (mM) (hr) CS Chain 10 24 3 20 24 7.2 30 24
8.5 20 48 9 30 48 10.5
[0368] The concentration of CS was kept constant at 20 mg per mL
for all oxidation reactions. The measured amounts of CS and sodium
periodate were reacted and protected from light in 0.1 M sodium
acetate buffer (pH 5.5) for the durations specified. Completion of
the reaction was obtained by removing sodium periodate by
performing gel filtration chromatography with a Bio-Scale Mini
Bio-Gel column packed with polyacrylamide beads (Bio-Rad
Laboratories, Hercules, Calif.) using an AKTA Purifier FPLC (GE
Healthcare, Piscataway, N.J.). The running buffer used for the
desalting process was 1.times. Phosphate Buffered Saline (PBS, pH
7.4, Invitrogen, Carlsbad, Calif.).
[0369] N-[.beta.-Maleimidopropionic acid]hydrazide,trifluoroacetic
acid salt (BMPH, Pierce, Rockford, Ill.) was reacted in a 50 M
excess with the desalted, oxidized CS in 1.times. PBS. The
hydrazide end of BMPH reacts to covalently attach to the
functionalized CS, via the newly created aldehydes, to form a
Schiff base intermediate. Sodium cyanoborohydride (5 M, Pierce) was
added to the reaction to reduce the Schiff base intermediate imine
to a more stable amine. Excess BMPH was removed from the solution
by FPLC desalting in deionized water. Due to the absorbance
detection capabilities on the AKTA Purifier FPLC, the amount of
excess BMPH was measured. The small size and low molecular weight
of BMPH (297.19 g/mol) resulted in its elution from the column at a
separate, much later timepoint. With the presence of its numerous
single bonds and occasional double bonds, BMPH produced a strong
absorbance spectrum at both the 215 nm wavelength (characteristic
of single bonds) and 254 nm wavelength (characteristic of double
bonds). Therefore, a standard curve was produced, correlating known
BMPH masses to the integrated area of the 215 nm absorbance
spectra, FIG. 2. With this standard curve, the mass of excess BMPH
was determined. Subtracting the excess BMPH mass from the original
reaction mass allows the determination of the mass of BMPH consumed
in the reaction. Using the consumed mass, the number of BMPH bound
to the oxidized CS was calculated. The collected CS-BMPH product
was frozen, lyophilized, and stored at -80.degree. Celsius.
[0370] The HA binding peptide sequence was identified by Mummert.
Slight modifications to the identified sequence produced the
specific HA binding sequence, GAHWQFNALTVRGGGC (noted as GAH), that
was used in this research. The peptide was produced by and
purchased from Genscript (Piscataway, N.J.). The cysteine amino
acid was included to allow coupling, by way of thioether bond
formation, to the maleimide group of BMPH. This reaction occurs at
a 1:1 ratio, allowing the assumption that the number of BMPH bound
to the functionalized CS will equal the number of GAH peptides
attached. GAH peptide, at one molar excess to the number of BMPH
coupled per chain, was dissolved in dimethyl sulfoxide (DMSO,
Sigma) and was added to the CS-BMPH solution in 15 minute
intervals, a quarter of the volume at a time. After the last
addition of GAH peptide, the reaction was allowed to progress for
two hours. During this time, the excess GAH peptide formed
particulates. Before purifying the solution to obtain GAH
functionalized CS, the solution was passed through an Acrodisc 0.8
.mu.m pore diameter filter (Pall, Port Washington, N.Y.) to remove
the excess peptide particulates. The solution was then passed, with
deionized water, through the AKTA Purifier FPLC to purify the
GAH-CS compound. The collected compounds were then frozen at
-80.degree. Celsius and lyophilized to produce the desired
aggrecan-mimics. By laboratory convention, the aggrecan mimic was
named by (# of peptides attached) (first three letters of peptide
sequence)--(GAG abbreviation that was functionalized) i.e. for the
aggrecan mimic, 3GAH-CS for 3 GAH HA binding peptides
functionalized to a chondroitin sulfate GAG backbone.
EXAMPLE 3
Binding of Synthetic Peptidoglycan to Hyaluronic Acid
Synthetic Peptidoglycan Binding to Immobilized Hyaluronic Acid
[0371] Hyaluronic Acid (HA, from Streptococcus equi, Sigma) at a
concentration of 4 mg per mL, was immobilized to a 96-well plate
(Costar, blk/clr, Corning, Corning, N.Y.) overnight at 4.degree.
Celsius. Biotin labeled GAH peptides were bound, by way of BMPH, to
functionalized CS at a concentration of 1 biotin-GAH per CS chain.
Unlabeled GAH peptides bound to the remaining unreacted aldehydes
of CS. Standard biotin-streptavidin detection methods were utilized
to determine the degree of aggrecan mimic binding to the
immobilized HA. Blocking of the HA surface was done for one hour
with 1% Bovine Serum Album (BSA, Sera Care Life Sciences, Milford,
Mass.) in 1.times. PBS solution. After washing with 1.times. PBS,
the biotin-labeled aggrecan mimic was incubated in the well for 30
minutes and then washed with 1.times. PBS. Streptavidin-horseradish
peroxidase (R&D Systems, Minneapolis, Minn.) solution was added
to each well, and allowed to react for 20 minutes. After reaction
completion and washing, chromogen solution was added (Substrate
Reagent Pack, R&D Systems) and developed for 15 min. At 15 min,
sulfuric acid (Sigma) was added directly to each well to stop the
reaction. The well plate was then read on the M5 SpectraMax Plate
Reader (Molecular Devices, Sunnyvale, Calif.) at 450 and 540 nm
wavelengths. By subtracting the two absorbance readings produced,
the absorbance due to the bound biotin-labeled aggrecan-mimic was
determined.
[0372] One GAH peptide per aggrecan mimic was replaced by a
biotin-labeled GAH peptide and the now-labeled aggrecan mimic was
incubated with immobilized HA. Commercially available biotin
detection products (through streptavidin and HRP) demonstrated the
degree of mimic binding to the immobilized HA (see FIG. 3).
Starting at a concentration of 1 .mu.M, the aggrecan mimic had a
dose dependent increase in presence on the immobilized HA, proving
that the mimic was binding to the HA. However, the determination of
the mimic's binding affinity was not pursued due to the uncertainty
of the amount of HA immobilized.
Rheometer Derived Synthetic Peptidoglycan Binding to Hyaluronic
Acid
[0373] HA solutions were created to test the aggrecan-mimic's
ability to bind to HA in a more physiologically relevant situation.
The ability of the aggrecan-mimic to bind to HA was deduced by the
improvement in storage modulus of the solution, indicating HA
crosslinking by the mimic. Multiple treatments were created in
1.times. PBS pH 7.4 to test the aggrecan mimic's ability to bind
HA: 2.5 wt % HA control, HA+CS at a 25:1 molar ratio of CS:HA,
HA+3GAH-CS at 25:1, HA+7.2GAH-CS at 25:1, HA+10.5GAH-CS at
25:1.
[0374] Using the AR-G2 Rheometer (TA Instruments, New Castle,
Del.), frequency (0.1-100 Hz, 2.512 Pa) and stress (0.1-100 Pa, 1.0
Hz) sweeps were conducted to measure the storage modulus of each
solution.
[0375] Rheology studies the flow of a substance in response to
applied forces and is often used when measuring viscoelastic
materials. In particular, the rheometer determines the storage
modulus and the loss modulus based on the substance feedback to the
applied force. The storage modulus is a measure of the amount of
energy that is elastically absorbed by the substance and the loss
modulus depicts the amount of energy lost through heat. A large
storage modulus is indicative of a gel-like substance with a more
rigid, elastic structure; whereas, a small storage modulus and a
large loss modulus indicate a viscous material that does not
elastically retain the applied load. The high molecular weight HA
(.about.1 .5 MDa) is a very viscous material which elastically
retains a portion of the applied load due to a pseudo-gel formed by
HA chain entanglement. The created aggrecan mimic contains multiple
HA binding peptides which can act as a type of HA chain crosslinker
assuming adequate mimic binding to the HA. In solution with the
high molecular weight HA, it is hypothesized that the aggrecan
mimic could increase the rigidity of the solution, creating a
larger storage modulus. A larger storage modulus would be
indicative of extensive HA crosslinking, proving a strong binding
affinity between the aggrecan mimic and the HA chains present in
the mixture. Multiple versions of the aggrecan mimic were tested,
differentiated by the number of GAH peptides (on average either 3,
7.2, or 10.5) attached per functionalized CS chain.
[0376] The results of the experiment, shown in FIG. 4, showed that
the addition of CS significantly (.alpha.=0.05) lowered the storage
modulus of the HA solution. The addition of the dense negative
charges associated with the CS helped spread the HA chains, easing
the degree of HA entanglement and removing the pseudo-gel that
stored the applied energy. Confirming the hypothesis, as the number
of GAH peptides per CS increased from 3 to 10.5, the storage
modulus of the mixture increased as well. This increase can be
attributed to two beneficial attributes of having a higher number
of GAH peptides per aggrecan mimic. First, the more GAH peptides
attached per CS, the higher the avidity of the mimic, resulting in
a stronger mimic binding to the HA molecule. Second, the more GAH
peptides attached per CS, the greater the likelihood of the mimic
acting as a crosslinker between the HA molecules. Both effects
contributed to a more gel-like mixture, resulting in a larger
measured storage modulus. Weaker binding between the mimic and HA
would not restore the pseudo-gel and would be unable to store the
applied energy from the rheometer. The increase in storage modulus
confirms the strong mimic binding to the immobilized HA shown in
FIG. 3. Specifically at 10.5 GAH peptides per CS chain, the storage
modulus was significantly (.alpha.=0.05) higher than the HA+CS
control, reaching an average storage modulus similar to the HA
control.
EXAMPLE 4
Synthetic Peptidoglycan Compression Studies
Collagen Gel Formation and Turbidity
[0377] To mimic the native cartilage extracellular matrix, collagen
was utilized to entrap the HA and aggrecan-mimic aggregates within
a natural scaffold. Collagen type II (CII) was obtained from two
different commercial sources (Affymetrix, Santa Clara, Calif. and
Sigma). Mixtures of the cartilage ECM components were prepared in
TES Buffer (60 mM TES, 20 mM Na.sub.2HPO.sub.4, 0.56 M NaCl,
chemicals from Sigma) pH 7.6 according to the native component
breakdown, where CII comprised 70 dry wt % and the combination of
HA and the aggrecan mimic/CS control formed the remaining 30 dry wt
% of the mixture. The final concentration of CII in the gel was 2
mg per ml. Samples consisted of a CII control, CII+HA+CS control,
and CII+HA+aggrecan mimic (10.5 GAH-CS). To prevent premature
fibrillogenesis and gel formation, the solutions were kept on ice
at an acidic pH. Solution mixtures of the components were placed in
a 384 well plate (Greinier blk/clr, Monroe, N.C.), placed at
37.degree. C. and physiological pH to initiate fibrillogenesis, and
were monitored at 313 nm on the M5 SpectraMax to determine gel
formation. CII was unable to form gels when included with the
varying treatments (See Supplementary Information). Therefore,
collagen type I (CI, High Concentration Rat Tail Collagen Type 1,
BD Biosciences, Bedford, Mass.) was utilized for the gel formation.
The same treatments and procedure were used with the CI, except
that the component masses were shifted for a CI final concentration
of 4 mg per mL. CI was used for all following experiments.
[0378] Turbidity with CI was performed to measure the formation of
the cartilage replicate, results shown in FIG. 5. As demonstrated,
the addition of HA+10.5 GAH-CS did not affect the fibrillogenesis
of the collagen fibers. All treatments followed a similar curve and
reached similar absorbance peaks at about the same time. HA+10.5
GAH-CS treatment had a higher initial absorbance due to the
aggrecan mimics tendency to form self-aggregates in 1.times. PBS
solution, not due to premature CI fibril formation. The aggregation
of 10.5 GAH-CS was recognized during the initial HA rheometer
tests, but the aggregation did not inhibit the aggrecan mimic's
ability to bind to HA.
Collagen Gel Property Testing
[0379] Collagen-based gel compression tests and frequency sweeps
were conducted using an AR-G2 Rheometer using a 20-millimeter
parallel plate geometry (TA Instruments). The 375 .mu.L gel
mixtures were prepared on ice and pipetted onto the rheometer base
plate. The geometry was lowered to a gap distance of 1 mm and the
solution was heated to 37.degree. Celsius. A humidity trap was
utilized to prevent gel dehydration while the mixture was allowed
to gel over two hours. This two hour value was determined by the
demonstrated time to gelation data from the turbidity data. After
the two hour time period, the gels were compressed or oscillated
depending on the test. Compression tests occurred at an engineering
strain rate of 1% (10 .mu.m) per second. The gap distance and the
normal force on the geometry head were measured. The frequency
sweeps measured the storage modulus of the created gels during a
logarithmic base ten increase in frequency from 0.1 to 1 Hz.
[0380] The simultaneous normal force and displacement were
measured, and the engineering stress and strain were calculated for
the treatments. As shown in FIG. 6, the inclusion of the aggrecan
mimic significantly (.alpha.=0.05) increased the compressive
strength of the gel complex. The peak engineering stress of the
collagen+HA+AGG mimic reached 7.5 kPa at an engineering strain of
9%, whereas the collagen+HA+CS control reached a peak of 4.8 kPa at
4%, and the collagen control reached a peak of 4.2 kPa at 15%
strain.
[0381] Two factors contributed to the increase in compressive
strength of the CI+HA+10.5 gel, the first being the mimic's ability
to attract water and the second being the HA crosslinking ability
of the aggrecan mimic. In native cartilage, the predominance of the
entrapped negative charges provided by the HA and CS attract water
and retard its diffusion from the cartilage. When a compressive
force is applied to the cartilage, the water is not able to diffuse
out into the synovial capsule. Retaining this incompressible water
increases the compressive strength of the structure. Similarly in
the tested gel complexes, the inclusion of the negative charges
associated with CS in the gel provides the same attraction. As can
be seen in FIG. 6, both the CS and 10.5 GAH-CS treatments have an
increased compressive strength. The CS treatment is not fixed
within the CI complex (it is not bound to HA) and therefore after a
small compressive deformation, the CS and its attracted water
diffuse out of the complex into the surrounding fluid. The
diffusion of the CS and water from the complex diminishes the
compressive strength of the complex, causing the resulting gel's
compressive profile to resemble that of the collagen scaffold
control. In contrast, 10.5 GAH-CS is bound to the interwoven HA.
Therefore, a much higher compressive stress is required to overcome
the binding of the mimic to HA and cause the diffusion of CS and
attracted water from the complex.
[0382] Secondly, the ability of the aggrecan mimic to act as a HA
crosslinker results in a higher degree of entrapment for the HA and
mimic. Effectively, the HA crosslinking nature creates large
aggregates within the collagen complex, similar to the native
aggrecan/HA aggregates. The main difference between the aggrecan
mimic and native aggrecan is the size of the molecule. The protein
backbone of aggrecan alone weighs .about.220 kDa, whereas the
aggrecan mimic, in entirety, only weighs around 30 kDa. Therefore,
the native aggregate complex, with over 100 aggrecan molecules
bound to the HA, produces much larger aggregates than the aggrecan
mimic could produce. However, by acting as a crosslinker between HA
chains, the aggrecan mimic can produce its own form of an aggregate
that also portrays the main characteristics of native aggregates;
voluminous, negatively-charged structures. The role of the aggrecan
mimic as an HA crosslinker was further investigated by applying
shear loads through rheo logical tests on the CI gels described
above. The results of these experiments can be seen in FIG. 7.
[0383] The inclusion of 10.5 GAH-CS significantly (.alpha.=0.05)
increased the storage modulus of the formed gel. The network
created by the binding of the mimic to the HA supplemented the
existing rigidity of the CI matrix, allowing an increased elastic
absorbance of the energy applied by shear loading. This study was
important as it verified the crosslinking ability of the 10.5
GAH-CS and the creation of an alternate aggregate form.
EXAMPLE 5
Synthetic Peptidoglycan Protection of Hyaluronic Acid
Degradation
[0384] Dynamic viscosity values of HA solutions were determined
using the AR-G2. High molecular weight HA solutions have a large
viscosity due to the extensive chain entanglement caused by the
long chain length. Hyaluronidase (Type II from Sheep Testes, Sigma)
cleaves the HA chain, creating shorter chains with less
entanglement. The shorter HA chains will have a measurably lower
viscosity. HA solutions were incubated with 100 units/mL
hyaluronidase. Dynamic viscosities were determined using a time
sweep with constant angular frequency and oscillatory stress
initially and at 2 and 4-hour timepoints. Samples (at 0.5 wt % HA)
consisted of HA, HA+CS, and HA+10.5 GAH-CS. The treatment values
were added at a 75:1 treatment to HA molar ratio. The percent
degradation was calculated for each measurement by dividing the
initial viscosity from the difference of the measured viscosity
minus the initial viscosity.
[0385] Work by Pratta et al. and Little et al. has shown the
importance of aggrecan in preventing cartilage component
degradation. The demolition of the cartilage matrix in
osteoarthritis is started with the cleavage of the aggrecan
proteoglycans. The removal of the
[0386] GAG-rich region of the proteoglycan exposes the remaining
components, CII and HA, to degrading enzymes. With the knowledge of
the importance of aggrecan in preventing degradation, studies were
conducted to determine the ability of the aggrecan-mimic in
preventing HA degradation.
[0387] The viscosity of a HA solution is dependent on the size of
the HA chains. Due to entanglement, larger HA chains will produce a
higher viscosity. When exposed to hyaluronidase, the HA chain is
cleaved into smaller units. Therefore, the size of the HA and the
amount of HA entanglement decreases. This decrease prompts a
similar decrease in the measured viscosity. The percent change in
viscosity of HA solutions in the presence of hyaluronidase will
provide key information into the amount of degradation the HA has
undergone. FIG. 8 presents the percent degradation of HA control
versus the associated treatments. As can be seen, the AGG mimic,
GAH, significantly reduced the rate of degradation of HA, indiating
that it behaves similarly to native AGG in its protection of ECM
components.
[0388] Viscosities of each treatment without hyaluronidase (TES
Buffer replaced the hyaluronidase volume) were initially measured
and served as the baseline for the percent degradation
calculations. The 0 hr timepoint involved the addition of the
hyaluronidase, mixing of the solution, pipetting onto the
rheometer, and the beginning equilibration operation of the machine
Therefore, the 0 hr timepoint occurred approximately two minutes
after the addition of hyaluronidase. A high concentration of
hyaluronidase (25 units per mL) was utilized to replicate the worst
possible scenario. In addition, the HA molecules were dispersed in
solution, rather than tightly interwoven into a collagen network.
As can be seen from FIG. 8, both the HA Control and the HA+CS
treatment had almost complete degradation of the HA solution at the
0 hr timepoint. In contrast, the addition of 10.5 GAH-CS
significantly (.alpha.=0.05) reduced the amount of HA degradation.
In fact, the presence of 10.5 GAH-CS increased the viscosity above
the baseline values. It is believed that the addition of
hyaluronidase cleaves some of the excess HA. This allows 10.5GAH-CS
to better crosslink the remaining, intact chains, creating a denser
gel which produced the larger viscosity. At the 2 hr timepoint,
both the HA control and HA+CS had completely degraded with percent
degradations above 90%, but the HA solution with 10.5 GAH-CS had a
significantly (.alpha.=0.05) lower percent degradation. Lastly, at
the 4 hr timepoint, all treatments had been degraded, with their
percent degradations all above 90%. Amongst the three timepoints,
10.5 GAH-CS was not able to completely prevent HA degradation, but
it drastically reduced the rate of degradation compared to the
degradations of the HA Control and HA+CS. This reduced rate
demonstrates that the 10.5 GAH-CS prevents the degradation of the
HA chains. It is believed that this prevention is being
accomplished through competitive inhibition of the hyaluronidase
cleavage point on the HA chain. The non-covalent binding of the
mimic to the HA chain coupled with the gradual degradation rate of
the HA chains appear to validate this belief. In addition, the
degradation rate of the 10.5 GAH-CS solution is still believed to
be artificially high. Upon incubation of the mimic within the HA
solution, HA+10.5 GAH-CS aggregates were formed. However, these
aggregates did not spread uniformly throughout the solution volume.
Therefore, the solutions were mixed, similarly to the other
samples, before a measurement was taken. The mixing of the solution
disrupted the aggregates, dislodging 10.5 GAH-CS and exposing the
hyaluronidase cleavage point. Even after the 4 hr timepoint, when
supposedly complete degradation had occurred, substantial
aggregation of HA+10.5 GAH-CS still occurred. In a compact matrix
like the ECM of cartilage, it is possible that 10.5 GAH-CS could
not only significantly reduce the degradation rate, but suppress HA
degradation.
EXAMPLE 6
CryoScanning Electron Microscopy (SEM)
[0389] The ECM-based constructs, as described for turbidity
measurements, were formed on an SEM plate at 37.degree. C.
overnight. The SEM plates were secured into a holder, and were
plunged into a liquid nitrogen slush. A vacuum was pulled on the
sample as it was transferred to the Gatan Alto 2500 pre-chamber.
Within the chamber, cooled to -170.degree. C., a cooled scalpel was
used to create a free break surface on the sample. The sample was
subjugated to sublimation at -85.degree. C. for 15 minutes followed
by a sputter-coating of platinum for 120 seconds. After
sputter-coating, the sample was transferred to the microscope stage
and images were taken at -130.degree. C.
[0390] Representative images were obtained at a magnification of
10,000.times., as shown in FIG. 9. Panel A shows the CI control,
and is characterized by extensive crosslinking between major
fibrils, and relatively small matrix pore size. Panel B shows
CI+HA+CS, and contains extensive crosslinking, but larger pore
size, due to the presence of the large HA chains. Panel C shows
CI+HA+10.5 GAH-CS and illustrates a noticeably smaller degree of
crosslinks in addition to a very large pore size. The AGG mimic can
bind to the HA creating a relatively large, cumbersome complex that
hinders the CI crosslinking
[0391] As can be qualified in the representative images, the
addition of HA+CS did not have an effect on the variation of
collagen fibril diameters, but the HA+CS sample did have a larger
representative void space. In comparison to the control groups, the
addition of the AGG mimic with the HA resulted in a smaller
variation of collagen fibril diameters due to the limited number of
small fibril diameters, and an overall increase in the void space
of the sample. The binding of the AGG mimic to the HA molecule
created an aggregate complex that was trapped within the collagen
scaffold and excluded smaller fibril formation between the larger
fibrils due to steric hindrance.
EXAMPLE 7
Collagen Protection
[0392] ECM-based constructs containing collagen alone,
collagen+HA+CS, or collagen+HA+10.5 GAH-CS were created in 8-well
chambered slides as described previously. The final sample volume
was 200 .mu.L consisting of 0.8 mg of collagen type I. Matrix
metalloprotease-I (MMP-I, R&D Systems, Minneapolis, Minn.) at a
concentration of 0.133 mg/mL, was activated following the protocol
detailed in the manufacturer's instructions. Briefly, MMP-1,
already dissolved in manufacturer's buffer (50 mM Tris, 10 mM
CaCl.sub.2, 150 mM NaCl, 0.05% Brij-35, pH 7.5), was combined with
an equal volume of 25 mM APMA (Sigma) in DMSO at 37.degree. C. for
2 hrs to activate the enzyme. Upon activation, the MMP-1 solution
was diluted two fold in water and was added to the sample as a 100
.mu.L supernatant. The samples were incubated at 37.degree. C. with
gentle shaking Twenty-five hrs after the addition of the initial
enzyme solution, the supernatant was removed and replaced with a
fresh batch of enzyme. After 50 total hr of incubation with the
enzyme, the remaining gels were removed from the chambered slides,
washed with deionized water to remove any enzyme solution or
degradation products, and resolubilized in 12 M HCl. The samples
were diluted in water to reach a final concentration of 6 M HCl,
and were hydrolyzed overnight at 110.degree. C. Following
hydrolysis, the amount of hydroxyproline (hyp) was analyzed
according to the protocol developed by Reddy, et al. (Clin Biochem,
1996, 29: 225-9). Briefly, the hydrolyzed samples were incubated
with Cholramine T solution (0.56 M) for 25 minutes at room
temperature before the addition of Elrich's reagent and subsequent
chlorophore development for 20 minutes at 65.degree. C. After the
development of the chlorophore, the samples were read on a
spectrophotometer at a wavelength of 550 nm. Absorbance readings
were compared to those obtained from known concentrations of
collagen to determine the amount of collagen remaining in each
sample.
[0393] Each replicate sample was constructed with 0.8 mg of CI, and
after degradation, the remaining CI amount was determined by the
protocol developed by Reddy et al. and converting that to CI amount
by a set of CI standards. The percent degradation was determined by
subtracting the remaining CI from the initial CI, dividing by the
initial CI, and multiplying by 100. The percent degradation of the
three treatments is shown in FIG. 10. All the treatments were
significantly different from each other (p<0.05). In particular,
the percent degradation of the AGG mimic sample (CI+HA+10.5 GAH-CS
=41.0%) was significantly less (p<0.05) than the other two
treatments (CI=64.5% and CI+HA+CS=74.7%). The presence of the AGG
mimic significantly reduced the CI degradation. The presence of the
AGG mimic can act as a hindrance to the cleavage sites of the
degrading enzymes. By creating the large aggregates with HA that
are tightly trapped within the collagen scaffold, the AGG mimic can
occupy the space proximal to the collagen, preventing enzyme access
to degradation locations.
EXAMPLE 8
Diffusion of Peptidoglycans Through Cartilage Matrix
[0394] Cartilage explants were obtained from the load bearing
region of three month old bovine knee joints. Native aggrecan was
removed from harvested cartilage explants leaving a matrix
consisting primarily of type II collagen and residual GAG. This was
achieved by treating explants with 0.5% (w/v) trypsin in HBSS for 3
hours at 37.degree. C. (FIG. 13). After trypsin treatment explants
were washed three times in HBSS and incubated with 20% FBS to
inactivate residual trypsin activity. Peptidoglycan was dissolved
in distilled water at 10 .mu.M concentration and diffused through
the articular surface of cartilage explants by placing 10 .mu.L of
the solution on the surface every ten minutes for one hour at room
temperature (FIG. 14). Normal cartilage and aggrecan depleted
cartilage were treated with 1.times. PBS as positive and negative
controls respectively. After diffusion, explants were washed three
times with 1.times. PBS and stored at -20.degree. C. until further
testing. Diffusion of peptidoglycan was confirmed by staining a
midsagittal section of the tissue with streptavidin-horseradish
peroxidase stain. The streptavidin stain binds to the biotin
labeled molecule and is depicted as a brown color (FIGS. 15 and
16).
EXAMPLE 9
Bulk Compression Testing
[0395] Displacement-controlled unconfined compression was performed
on an AR G2 rheometer with force transducers capable of detecting
normal forces in the range of 0.01-50 N (TA Instruments). The
explants were glued to the bottom of a hydrophobic printed
slide
[0396] (Tekdon) and covered in a 1.times. PBS bath. A 20 mm
diameter stainless steel parallel plate geometry head was lowered
until initial contact was made. Explant height was measured using a
digital micrometer (Duratool). Compressive loads from 0-30% nominal
strain (at 5% intervals) were applied to the explants through a
stepwise loading that involved a ramp duration of 5 sec (i.e. a
strain rate of 1.0%/sec) and hold time of 30 sec. Compressive
stiffness values were obtained by using the slope of equilibrium
stress values, computed during each hold section, versus respective
strain values, based on a linear fit model. Scaffolds tested for
bulk compression included: 1) Normal cartilage, 2) Aggrccan
depleted cartilage (AD), and 3) AD+mAGC (FIG. 17). Addition of the
HA binding peptidoglycan (mAGC) significantly restored stiffness of
cartilage explants to a higher extent as compared to the collagen
type II binding peptidoglycan (mAG(II)C).
EXAMPLE 10
Animal Model
[0397] Sprague-Dawley rats (250-300g) were used for surgery. The
patellar tendon, the anterior and posterior cruciate ligaments and
the medial, lateral collateral ligaments were transected. The
medial and lateral meniscuses were totally menisectomized. The knee
joint capsule was repaired with an absorbable suture and the skin
was closed with a 4-0 monofilament nylon. Starting at week 4, 10
.mu.l of a 1 .mu.m mAGC was administered weekly.
[0398] The extent of inflammation was indicated by the MMP-13 probe
(FIG. 18) in Sprague-Dawley rats treated with and without
peptidoglycan at four, six and eight weeks post surgery (FIG. 19).
X-ray images of Sprague-Dawley rat knee joints showed injured knee
6 weeks and 8 weeks following OA induction (FIG. 20, Panels A and
D, respectively), injured knee with peptidoglycan treatment (FIG.
20, Panels B and E, respectively), and normal knee (FIG. 20, Panel
C) six weeks after osteoarthritis induction surgery. MicroCT of
Sprague-Dawley rats indicated re-growth of new cartilage six and
eight weeks after OA induction surgery. Injured knees 6 weeks and 8
weeks following OA induction, (FIG. 21, Panels A and D,
respectively), injured knees following peptidoglycan treatment
(FIG. 21, Panels B and E, respectively), and Normal knee (FIG. 21,
Panel C), are shown.
EXAMPLE 11
Reagents
[0399] Peptide GAHWQFNALTVRGGGC (GAH) was purchased from Genscript
(Piscataway, N.J.). N-[.beta.-maleimidopropionic acid] hydrazide,
trifluoroacetic acid salt (BMPH) was purchased from Pierce
(Rockford, Ill.). Rat tail type I collagen was purchased from BD
Biosciences (Bedford, Mass.). Human recombinant interlukin-1.beta.
was purchased from Peprotech (Rocky Hill, N.J.). All other supplies
were purchased from VWR (West Chester, Pa.) or Sigma-Aldrich (St.
Louis, Mo.) unless otherwise noted.
EXAMPLE 12
Collagen Scaffold Synthesis
[0400] Collagen scaffolds were prepared in TES buffer (60 mM TES,
20 mM Na.sub.2PO.sub.4, 0.56 M NaCl) at a pH of 7.6. Scaffold
composition for mechanical testing and in vitro inflammatory model
studies are described in their respective sections. All solutions
were maintained on ice until fibrillogenesis was initiated at
37.degree. C. Aligned collagen scaffolds were created by placing
the collagen solution at the isocenter of a 9.4 Tesla magnet
(Chemagnetics CMX400) at 37.degree. C. for one hour, whereas
unaligned gels were prepared similarly but without magnetic
exposure. The slide containing the collagen solution was placed
parallel to the magnetic field, orienting the collagen fibers in a
direction perpendicular to the bottom of the slide. The gels were
then maintained at 37.degree. C. for 24 hours in a
humidity-controlled chamber to prevent evaporation.
EXAMPLE 13
Rheological Mechanical Testing
[0401] Shear and compression testing was performed on a
stress-controlled AR G2 rheometer (TA Instruments) using a 20 mm
diameter stainless steel parallel plate geometry head. Collagen
scaffolds were prepared on 20 mm diameter hydrophobic printed
slides (Tekdon). For shear tests, the geometry head was lowered
until contact was made at a gap height of 950 .mu.m. Preliminary
frequency and stress sweeps were performed to determine a linear
and stress-independent storage modulus range. Frequency sweeps were
then performed on all gels with an oscillatory stress of 0.2 Pa
over a frequency range of 0.1 to 2 Hz. For compression tests, the
geometry head was lowered until contact was made with the scaffold
at a gap height of 1000 .mu.m. Compressive loads from 0-30% nominal
strain (at 5% intervals) were applied to the collagen scaffold
through a stepwise loading that involved a ramp duration of 5 sec
(i.e. a strain rate of 1.0%/sec) and hold time of 30 sec.
Compressive stiffness values were obtained by using the slope of
equilibrium stress values, computed during each hold section,
versus respective strain values, based on a linear fit model.
Collagen scaffold composition for mechanical tests were: 1)
Unaligned collagen, 2) Aligned collagen, 3) Unaligned collagen+mAGC
and 4) Aligned collagen+mAGC.
[0402] Bulk Mechanical Analysis: The aggrecan mimic, mAGC, enhanced
bulk mechanical properties of scaffolds, irrespective of fiber
alignment (FIG. 22). For shear testing, the storage moduli values
at 0.5 Hz for unaligned and aligned collagen gels were 104.1.+-.3.6
Pa and 49.9.+-.5.4 Pa respectively. The addition of mAGC to the
collagen scaffold showed a significant increase in the storage
moduli of the unaligned and aligned gels to 113.9.+-.4.6 Pa and
76.6.+-.3.6 Pa respectively (p<0.001). Unaligned gels showed a
higher storage modulus as compared to aligned gels (p<0.0001).
For compression testing, the compressive stiffness for aligned
scaffolds (2478.+-.250 Pa) was lower than unaligned scaffolds
(3564.+-.315 Pa) (p<0.001). Addition of mAGC to these scaffold
systems increased compressive stiffness of the aligned and
unaligned scaffolds to 4626.+-.385 Pa and 5747.+-.306 Pa,
respectively (p<0.0001).
EXAMPLE 14
In Vitro Inflammation Model
[0403] Collagen scaffolds seeded with chondrocytes were stimulated
with IL-1.beta. and assessed for degradation products.
[0404] Chondrocyte Isolation: Primary chondrocytes were harvested
from three-month-old bovine knee joints obtained from an abattoir
within 24 hours of slaughter (Dutch Valley Veal). Cartilage slices,
150-200 .mu.m thick, were shaved from the lateral femoral condyle
and washed three times in serum-free DMEM/F-12 medium (50 .mu.g/mL
ascorbic acid 2-phosphate, 100 .mu.g/mL sodium pyruvate, 0.1%
bovine serum albumin, 100 units/mL penicillin, 100 .mu.g/mL
streptomycin and 25 mM HEPES) prior to digestion with 3% fetal
bovine serum (FBS) and 0.2% collagenase-P (Roche Pharmaceuticals)
at 37.degree. C. for six hours. Released chondrocytes were filtered
through 70 .mu.m cell strainer and centrifuged at 1000 rpm three
times for five minutes each in medium listed above supplemented
with 10% FBS. The cell pellet was resuspended in 10% FBS
supplemented media and plated on 10 cm dishes at 10,000 cells/mL in
a 37.degree. C., 5% CO.sub.2 humidified incubator until
confluent.
[0405] Scaffold Fabrication: Upon reaching confluency, cells were
trypsinized and encapsulated at 10,000 cells/mL within collagen
scaffolds (Table 3) and allowed to equilibrate for 3 days prior to
treatment.
TABLE-US-00018 TABLE 3 Scaffold composition for in vitro testing
Unaligned Collagen Experimental Setup A: Collagen + CS + HA +
IL-1.beta. B: Collagen + CS + HA C: Collagen + mAGC + HA +
IL-1.beta. D: Collagen + mAGC + HA Aligned Collagen Experimental
Setup E: Collagen + CS + HA + IL-1.beta. F: Collagen + CS + HA G:
Collagen + mAGC + HA + IL-1.beta. H: Collagen + mAGC + HA
[0406] Inflammation Model: Constructs were incubated with or
without 20 ng/mL IL-1.beta. in chemically-defined media
supplemented with 5% FBS and antibiotics (100 units/mL penicillin
and 100 .mu.g/mL streptomycin). Culture medium was replaced every
two days. Removed media extracts were stored at -80.degree. C.
until further testing.
[0407] Degradation Assay: GAG degradation was monitored by
measuring CS released in cell culture media using the
dimethylmethylene blue (DMMB) dye assay and computed with a
chondroitin-6-sulfate standard curve. Similarly, type I collagen
degradation in cell culture media was monitored using the Sircol
Collagen Assay using manufacturer specified protocols (Bio-Color).
GAG and collagen degradation were reported as cumulative release
over an eight-day culture period.
[0408] Proteolytic Degradation Analysis: The amount of CS and
collagen released into cell culture media was significantly
decreased when scaffolds that contained mAGC (FIGS. 11, 12, 23 and
24) (p.sub.cs<0.001 and p.sub.collagen<0.02, respectively).
Aligned collagen gels showed a statistically higher CS and collagen
release into the media as compared to unaligned collagen fibers
(p<0.001).
[0409] As described herein, the hyaluronic-binding synthetic
peptidoglycan is able to protect HA and the underlying collagen
fibers in the scaffold from proteolytic cleavage. The synthesis of
the hyaluronic-binding synthetic peptidoglycan utilized the
chondroprotective benefits of CS. CS has been shown to
down-regulate matrix metalloproteases production. Our synthetic
peptidoglycan design herein described allowed CS chains to be
attached to HA, preventing degradation of both molecules. By
placing the synthetic peptidoglycan in an environment rich in
proteolytic enzymes, its ability to prevent excessive loss of ECM
components has been demonstrated.
EXAMPLE 15
Real-time PCR
[0410] Following the cell culture study, constructs were stored in
RNAlater solution (Ambion) at 4.degree. C. for less than one week.
Total mRNA was extracted using Nucleospin RNA II (Clontech)
according to manufacturer's protocols. Extracted mRNA from all
samples was quantified using Nanodrop 2000 spectrophotometer
(Thermo Scientific) and reverse transcribed into cDNA using High
Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems).
Real-time PCR was performed using Taqman Gene Expression Assays
(Applied Biosystems) with the following primers: GAPDH
(Bt03210913_gl), aggrecan (Bt03212186_ml) and collagen type II
(Bt03251861_ml). 60 ng of cDNA template was prepared per 20 .mu.L
reaction for the two genes of interest and the endogenous gene.
Real-time PCR analysis was carried out using a Taqman PCR Master
Mix and 7500 Real-Time PCR System (Applied Biosystems). Data
reported was normalized to GAPDH gene expression.
[0411] mRNA Expression Analysis: Collagen alignment, presence of
aggrecan mimic and stimulation with IL-1.beta. significantly
effected aggrecan (p.sub.alignment<0.001,
p.sub.peptidoglycan<0.02 and p.sub.IL-1.beta.<0.001) and
collagen type II expression (p.sub.alignment<0.01,
p.sub.peptidoglycan<0.001 and p.sub.IL-1.beta.<0.015). The
presence of mAGC limited excessive loss of CS from the scaffold,
which results in a lower aggrecan expression (p<0.02) (FIG. 25).
The presence of mAGC also limited collagen degradation. However,
collagen type II expression depended on the extent of collagen lost
during degradation (FIG. 25). In unaligned scaffolds, the level of
collagen type II expression was higher in scaffolds prepared
without mAGC, whereas in aligned collagen scaffolds, the level of
collagen type II was higher in scaffolds prepared with mAGC
(p<0.05).
EXAMPLE 16
Statistical Analysis
[0412] Each experiment was repeated twice, with at least n=3 in
each data set. Statistical significance for mechanical test data
was analyzed with a two-way ANOVA with alignment and addition of
peptidoglycan as factors. The cell culture data was analyzed using
a three-way ANOVA with alignment, addition of peptidoglycan, and
treatment with IL-1.beta. as factors. A post-hoc Tukey pairwise
comparison (.alpha.=0.05) was used to directly compare scaffolds
prepared with and without the aggrecan mimic in each system.
Sequence CWU 1
1
59110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Gly Ala His Trp Gln Phe Asn Ala Leu Thr Val Arg
Gly Gly 1 5 10 312PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 3Gly Asp Arg Arg Arg Arg Arg Met Trp His
Arg Gln 1 5 10 412PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 4Gly Lys His Leu Gly Gly Lys His Arg Arg
Ser Arg 1 5 10 511PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Arg Gly Thr His His Ala Gln Lys Arg Arg
Ser 1 5 10 612PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 6Arg Arg His Lys Ser Gly His Ile Gln Gly
Ser Lys 1 5 10 712PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 7Ser Arg Met His Gly Arg Val Arg Gly Arg
His Glu 1 5 10 812PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 8Arg Arg Arg Ala Gly Leu Thr Ala Gly Arg
Pro Arg 1 5 10 912PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 9Arg Tyr Gly Gly His Arg Thr Ser Arg Lys
Trp Val 1 5 10 1012PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 10Arg Ser Ala Arg Tyr Gly His Arg Arg
Gly Val Gly 1 5 10 1112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Gly Leu Arg Gly Asn Arg Arg
Val Phe Ala Arg Pro 1 5 10 1212PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12Ser Arg Gly Gln Arg Gly Arg
Leu Gly Lys Thr Arg 1 5 10 1326PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 13Asp Arg Arg Gly Arg Ser Ser
Leu Pro Lys Leu Ala Gly Pro Val Glu 1 5 10 15 Phe Pro Asp Arg Lys
Ile Lys Gly Arg Arg 20 25 1412PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 14Arg Met Arg Arg Lys Gly Arg
Val Lys His Trp Gly 1 5 10 1512PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 15Arg Gly Gly Ala Arg Gly Arg
His Lys Thr Gly Arg 1 5 10 1626PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 16Thr Gly Ala Arg Gln Arg Gly
Leu Gln Gly Gly Trp Gly Pro Arg His 1 5 10 15 Leu Arg Gly Lys Asp
Gln Pro Pro Gly Arg 20 25 1712PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 17Arg Gln Arg Arg Arg Asp Leu
Thr Arg Val Glu Gly 1 5 10 1826PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 18Ser Thr Lys Asp His Asn Arg
Gly Arg Arg Asn Val Gly Pro Val Ser 1 5 10 15 Arg Ser Thr Leu Arg
Asp Pro Ile Arg Arg 20 25 1912PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Arg Arg Ile Gly His Gln Val
Gly Gly Arg Arg Asn 1 5 10 2012PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 20Arg Leu Glu Ser Arg Ala Ala
Gly Gln Arg Arg Ala 1 5 10 2112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Gly Gly Pro Arg Arg His Leu
Gly Arg Arg Gly His 1 5 10 2212PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Val Ser Lys Arg Gly His Arg
Arg Thr Ala His Glu 1 5 10 239PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 23Arg Gly Thr Arg Ser Gly Ser
Thr Arg 1 5 2412PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 24Arg Arg Arg Lys Lys Ile Gln Gly Arg
Ser Lys Arg 1 5 10 2510PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 25Arg Lys Ser Tyr Gly Lys Tyr
Gln Gly Arg 1 5 10 269PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 26Lys Asn Gly Arg Tyr Ser Ile
Ser Arg 1 5 279PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 27Arg Arg Arg Cys Gly Gln Lys Lys Lys 1
5 2811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Lys Gln Lys Ile Lys His Val Val Lys Leu Lys 1 5
10 2910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Lys Leu Lys Ser Gln Leu Val Lys Arg Lys 1 5 10
3010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Arg Tyr Pro Ile Ser Arg Pro Arg Lys Arg 1 5 10
319PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Lys Val Gly Lys Ser Pro Pro Val Arg 1 5
329PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Lys Thr Phe Gly Lys Met Lys Pro Arg 1 5
339PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Arg Ile Lys Trp Ser Arg Val Ser Lys 1 5
349PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Lys Arg Thr Met Arg Pro Thr Arg Arg 1 5
3516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Gly Ala His Trp Gln Phe Asn Ala Leu Thr Val Arg
Gly Gly Gly Cys 1 5 10 15 368PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 36Trp Tyr Arg Gly Arg Leu Gly
Cys 1 5 3712PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 37Arg Arg Ala Ser Arg Ser Arg Gly Gln
Val Gly Leu 1 5 10 3812PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 38Gly Arg Gly Thr His His Ala
Gln Lys Arg Arg Ser 1 5 10 3912PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Gln Pro Val Arg Arg Leu Gly
Thr Pro Val Val Gly 1 5 10 4012PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 40Ala Arg Arg Ala Glu Gly Lys
Thr Arg Met Leu Gln 1 5 10 4112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 41Pro Lys Val Arg Gly Arg Arg
His Gln Ala Ser Gly 1 5 10 4212PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Ser Asp Arg His Arg Arg Arg
Arg Glu Ala Asp Gly 1 5 10 4312PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 43Asn Gln Arg Val Arg Arg Val
Lys His Pro Pro Gly 1 5 10 4426PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 44Arg Glu Arg Arg Glu Arg His
Ala Val Ala Arg His Gly Pro Gly Leu 1 5 10 15 Glu Arg Asp Ala Arg
Asn Leu Ala Arg Arg 20 25 4526PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 45Thr Val Arg Pro Gly Gly Lys
Arg Gly Gly Gln Val Gly Pro Pro Ala 1 5 10 15 Gly Val Leu His Gly
Arg Arg Ala Arg Ser 20 25 4612PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 46Asn Val Arg Ser Arg Arg Gly
His Arg Met Asn Ser 1 5 10 4712PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 47Asp Arg Arg Arg Gly Arg Thr
Arg Asn Ile Gly Asn 1 5 10 4812PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Lys Thr Ala Gly His Gly Arg
Arg Trp Ser Arg Asn 1 5 10 4912PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 49Ala Lys Arg Gly Glu Gly Arg
Arg Glu Trp Pro Arg 1 5 10 5012PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 50Gly Gly Asp Arg Arg Lys Ala
His Lys Leu Gln Ala 1 5 10 5112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 51Arg Arg Gly Gly Arg Lys Trp
Gly Ser Phe Glu Gly 1 5 10 5213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 52Arg Asp Gly Thr Arg Tyr Val
Gln Lys Gly Glu Tyr Arg 1 5 10 5310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 53His
Arg Glu Ala Arg Ser Gly Lys Tyr Lys 1 5 10 5410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Pro
Asp Lys Lys His Lys Leu Tyr Gly Val 1 5 10 5510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Trp
Asp Lys Glu Arg Ser Arg Tyr Asp Val 1 5 10 5613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Lys
Gln Lys Ile Lys His Val Val Lys Leu Lys Gly Cys 1 5 10
5712PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 57Lys Leu Lys Ser Gln Leu Val Lys Arg Lys Gly Cys
1 5 10 586PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Trp Tyr Arg Gly Arg Leu 1 5 5910PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 59Gly
Pro Leu Gly Met Arg Gly Leu Gly Lys 1 5 10
* * * * *