U.S. patent application number 11/706922 was filed with the patent office on 2007-06-28 for functionalized derivatives of hyaluronic acid, formation of hydrogels in situ using same, and methods for making and using same.
This patent application is currently assigned to Orthogene LLC. Invention is credited to Daniel Aeschlimann, Paul Bulpitt.
Application Number | 20070149441 11/706922 |
Document ID | / |
Family ID | 22561268 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070149441 |
Kind Code |
A1 |
Aeschlimann; Daniel ; et
al. |
June 28, 2007 |
Functionalized derivatives of hyaluronic acid, formation of
hydrogels in situ using same, and methods for making and using
same
Abstract
Methods for chemical modification of hyaluronic acid, formation
of amine or aldehyde functionalized hyaluronic acid, and the
cross-linking thereof to form hydrogels are provided.
Functionalized hyaluronic acid hydrogels of this invention can be
polymerized in situ, are biodegradable, and can serve as a tissue
adhesive, a tissue separator, a drug delivery system, a matrix for
cell cultures, and a temporary scaffold for tissue
regeneration.
Inventors: |
Aeschlimann; Daniel;
(Madison, WI) ; Bulpitt; Paul; (Madison,
WI) |
Correspondence
Address: |
FISH & NEAVE IP GROUP;ROPES & GRAY LLP
1211 AVENUE OF THE AMERICAS
NEW YORK
NY
10036-8704
US
|
Assignee: |
Orthogene LLC
Sausalito
CA
|
Family ID: |
22561268 |
Appl. No.: |
11/706922 |
Filed: |
February 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10680000 |
Oct 6, 2003 |
7196180 |
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11706922 |
Feb 13, 2007 |
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09156829 |
Sep 18, 1998 |
6630457 |
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10680000 |
Oct 6, 2003 |
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Current U.S.
Class: |
536/18.7 ;
514/16.4; 514/17.1; 514/18.3; 514/19.3; 514/2.4; 514/3.3; 514/3.7;
514/54; 514/8.8; 514/8.9; 530/322; 536/53 |
Current CPC
Class: |
C08L 5/08 20130101; C12N
2533/80 20130101; C12N 5/0068 20130101; C08B 37/0072 20130101; A61L
24/08 20130101; A61K 9/0024 20130101; A61L 27/20 20130101; A61K
31/728 20130101; A61K 47/36 20130101; A61L 24/08 20130101; C08L
5/08 20130101; A61L 27/20 20130101; C08L 5/08 20130101 |
Class at
Publication: |
514/008 ;
514/054; 536/053; 530/322 |
International
Class: |
A61K 38/14 20060101
A61K038/14; A61K 31/728 20060101 A61K031/728; C08B 37/00 20060101
C08B037/00; C07K 9/00 20060101 C07K009/00 |
Claims
1. A composition comprising derivatives of hyaluronic acid (ha)
comprising disaccharide: subunits, wherein at least one of said
disaccharide subunits is a substituted disaccharide subunit having
a substitution at a carboxyl group, such that the substituted
disaccharide subunit is of the formula: ##STR23## wherein each of
R' and R'' is a side chain comprising one or more functional groups
selected from the group consisting of hydrogen; bioactive peptide;
alkyl; aryl; alkylaryl; arylalkyl; substituted alkylaryl containing
an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous;
substituted arylalkyl containing an atom or atoms of oxygen,
nitrogen, sulfur, phosphorous, halogen, or metal ion; and
substituted heterocycle containing an atom or atoms of oxygen,
nitrogen, sulfur, phosphorous, halogen or metal ion; wherein said
functional groups within or among each of said R' or R'' side
chains are either bound directly to each other or are separated by
a member selected from the group consisting of ether, keto, amino,
oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene;
and wherein each of said R' and R'' side chains terminates with a
terminal functional group selected from hydrogen, peptide,
aldehyde, amine, hydrazide, maleimide, sulfhydryl, active ester,
ester, carboxylate, imidoester, halogen and hydroxyl, wherein said
terminal functional groups of each of said R' and R'' side chains
may be bound directly to each other, with the proviso that when one
of R' or R'' is hydrogen, halogen or univalent metal ion, then R'
and R'' may not be bound directly to each other, and wherein said
derivatives of hyaluronic acid are covalently crosslinked via one
of said terminal functional groups.
2. The composition of claim 1, wherein at least one of said
terminal functional groups is selected from peptide, aldehyde,
amine, hydrazide, maleimide, sulfhydryl and active ester, whereby
said composition is amenable to crosslinking.
3. The composition of claim 1, wherein the molecular weight of said
composition is at least 100,000 daltons.
4. The composition of claim 1, wherein the molecular weight of said
composition is at most 100,000 daltons.
5. The composition of claim 1, wherein the molecular weight of said
composition is at least 1,000,000 daltons.
6. The composition of claim 1, wherein said composition is water
soluble.
7. A hydrogel of crosslinked HA derivatives, wherein said HA
derivatives are compositions according to claim 1.
8. The hydrogel of crosslinked HA derivatives of claim 7, wherein
said hydrogel is biodegradable.
9. A tissue adhesive comprising a hydrogel of claim 7, wherein the
side chain is selected from activated ester, aldehyde and
maleimide.
10. A tissue adhesive comprising a hydrogel of claim 7, wherein the
crosslinked HA derivatives are formed using a cross-linker selected
from polyvalent active ester, aldehyde and maleimide.
11. A tissue adhesive comprising a hydrogel of claim 7, wherein the
crosslinked hydrogel is formed in the presence of at least one
member selected from growth factors, cytokines, drugs, and
bioactive peptides.
12. The tissue adhesive of claim 11, wherein the crosslinked
hydrogel is formed in the presence of a growth factor and wherein
the growth factor is TGF-beta or BMP-2.
13. A matrix for cell cultures comprising the hydrogel of claim 7,
wherein the crosslinked HA-derivatives are formed using a
cross-linker selected from polyvalent active ester, aldehyde,
amine, arylazide, maleimide, and sulfhydryl.
14. A matrix for cell cultures comprising the hydrogel of claim 7,
wherein the crosslinked hydrogel is formed in the presence of at
least one member selected from growth factors, cytokines, drugs,
and bioactive peptides.
15. The matrix of claim 14, wherein the crosslinked hydrogel is
formed in the presence of a growth factor and wherein the growth
factor is TGF-beta or BMP-2.
16. A matrix for a scaffold comprising the hydrogel of claim 7,
wherein the crosslinked HA-derivatives are formed using a
cross-linker selected from polyvalent active ester, aldehyde,
amine, maleimide and sulfhydryl.
17. A matrix for a scaffold comprising the hydrogel of claim 7,
wherein the crosslinked hydrogel is formed in the presence of at
least one member selected from growth factors, cytokines, drugs,
and bioactive peptides.
18. The matrix of claim 17, wherein the crosslinked hydrogel is
formed in the presence of a growth factor and wherein the growth
factor is TGF-beta or BMP-2.
19. The matrix of claim 17, wherein the matrix further comprises
cells.
20. A method of forming a crosslinked biodegradable material under
physiological conditions from a composition comprising derivatives
of hyaluronic acid comprising disaccharide subunits, wherein at
least one of said disaccharide subunits is a substituted
disaccharide subunit having a substitution at a carboxyl group,
such that the substituted disaccharide subunit is of the formula:
##STR24## wherein each of R' and R'' is a side chain comprising one
or more functional groups selected from the group consisting of
hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl;
substituted alkylaryl containing an atom or atoms of oxygen,
nitrogen, sulfur, or phosphorous; substituted arylalkyl containing
an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen,
or metal ion; and substituted heterocycle containing an atom or
atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal
ion; wherein said functional groups within or among each of said R'
or R'' side chains are either bound directly to each other or are
separated by a member selected from the group consisting of ether,
keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne
and alkene; and wherein each of said R' and R'' side chains
terminates with a terminal functional group selected from the group
consisting of hydrogen, peptide, aldehyde, amine, arylazide,
hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate,
imidoester, halogen and hydroxyl; and wherein said derivatives of
hyaluronic acid are modified to an extent of more than 10%,
21. The method of claim 20, wherein the material is formed in
situ.
22. A method of regenerating tissue or causing tissue adhesion,
comprising contacting a tissue with a composition comprising
derivatives of hyaluronic acid comprising disaccharide subunits,
wherein at least one of said disaccharide subunits is a substituted
disaccharide subunit having a substitution at a carboxyl group,
such that the substituted disaccharide subunit is of the formula:
##STR25## wherein each of R' and R'' is a side chain comprising one
or more functional groups selected from the group consisting of
hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl;
substituted alkylaryl containing an atom or atoms of oxygen,
nitrogen, sulfur, or phosphorous; substituted arylalkyl containing
an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen,
or metal ion; and substituted heterocycle containing an atom or
atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal
ion; wherein said functional groups within or among each of said R'
or R'' side chains are either bound directly to each other or are
separated by a member selected from the group consisting of ether,
keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne
and alkene; and wherein each of said R' and R'' side chains
terminates with a terminal functional group selected from the group
consisting of hydrogen, peptide, aldehyde, amine, arylazide,
hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate,
imidoester, halogen and hydroxyl; and wherein said derivatives of
hyaluronic acid are modified to an extent of more than 10%.
23. The method of claim 22, wherein the method comprising one or
more of repairing cartilage, stemming hemorraging, reconstructing
nerves and/or vessels, and anchoring skin, vascular or cartilage
transplants or grafts.
24. A method of delivering cells or bioactive materials, comprising
delivering the cells or bioactive materials in a composition
comprising derivatives of hyaluronic acid comprising disaccharide
subunits, wherein at least one of said disaccharide subunits is a
substituted disaccharide subunit having a substitution at a
carboxyl group, such that the substituted disaccharide subunit is
of the formula: ##STR26## wherein each of R' and R'' is a side
chain comprising one or more functional groups selected from the
group consisting of hydrogen; bioactive peptide; alkyl; aryl;
alkylaryl; arylalkyl; substituted alkylaryl containing an atom or
atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted
arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur,
phosphorous, halogen, or metal ion; and substituted heterocycle
containing an atom or atoms of oxygen, nitrogen, sulfur,
phosphorous, halogen or metal ion; wherein said functional groups
within or among each of said R' or R'' side chains are either bound
directly to each other or are separated by a member selected from
the group consisting of ether, keto, amino, oxycarbonyl, sulfate,
sulfoxide, carboxamide, alkyne and alkene; and wherein each of said
R' and R'' side chains terminates with a terminal functional group
selected from the group consisting of hydrogen, peptide, aldehyde,
amine, arylazide, hydrazide, maleimide, sulfhydryl, active ester,
ester, carboxylate, imidoester, halogen and hydroxyl; and wherein
said derivatives of hyaluronic acid are modified to an extent of
more than 10%.
25. The method of claim 24, wherein the bioactive material is a
growth factor.
26. The method of claim 24, wherein the cells or bioactive material
are delivered to treat a pathological wound condition.
27. A method of generating tissue separation or preventing tissue
adhesions, comprising contacting tissue with a composition
comprising derivatives of hyaluronic acid comprising disaccharide
subunits, wherein at least one of said disaccharide subunits is a
substituted disaccharide subunit having a substitution at a
carboxyl group, such that the substituted disaccharide subunit is
of the formula: ##STR27## wherein each of R' and R'' is a side
chain comprising one or more functional groups selected from the
group consisting of hydrogen; bioactive peptide; alkyl; aryl;
alkylaryl; arylalkyl; substituted alkylaryl containing an atom or
atoms of oxygen, nitrogen, sulfur, or phosphorous; substituted
arylalkyl containing an atom or atoms of oxygen, nitrogen, sulfur,
phosphorous, halogen, or metal ion; and substituted heterocycle
containing an atom or atoms of oxygen, nitrogen, sulfur,
phosphorous, halogen or metal ion; wherein said functional groups
within or among each of said R' or R'' side chains are either bound
directly to each other or are separated by a member selected from
the group consisting of ether, keto, amino, oxycarbonyl, sulfate,
sulfoxide, carboxamide, alkyne and alkene; and wherein each of said
R' and R'' side chains terminates with a terminal functional group
selected from the group consisting of hydrogen, peptide, aldehyde,
amine, arylazide, hydrazide, maleimide, sulfhydryl, active ester,
ester, carboxylate, imidoester, halogen and hydroxyl; and wherein
said derivatives of hyaluronic acid are modified to an extent of
more than 10%,.
28. The method of claim 27, wherein the tissue separation or
prevention of tissue adhesions is effective for joint lubrication,
prevention of eye irritation or serving as a barrier to cells.
29. A method of augmenting tissue, comprising contacting tissue
with a composition comprising derivatives of hyaluronic acid
comprising disaccharide subunits, wherein at least one of said
disaccharide subunits is a substituted disaccharide subunit having
a substitution at a carboxyl group, such that the substituted
disaccharide subunit is of the formula: ##STR28## wherein each of
R' and R'' is a side chain comprising one or more functional groups
selected from the group consisting of hydrogen; bioactive peptide;
alkyl; aryl; alkylaryl; arylalkyl; substituted alkylaryl containing
an atom or atoms of oxygen, nitrogen, sulfur, or phosphorous;
substituted arylalkyl containing an atom or atoms of oxygen,
nitrogen, sulfur, phosphorous, halogen, or metal ion; and
substituted heterocycle containing an atom or atoms of oxygen,
nitrogen, sulfur, phosphorous, halogen or metal ion; wherein said
functional groups within or among each of said R' or R'' side
chains are either bound directly to each other or are separated by
a member selected from the group consisting of ether, keto, amino,
oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne and alkene;
and wherein each of said R' and R'' side chains terminates with a
terminal functional group selected from the group consisting of
hydrogen, peptide, aldehyde, amine, arylazide, hydrazide,
maleimide, sulfhydryl, active ester, ester, carboxylate,
imidoester, halogen and hydroxyl; and wherein said derivatives of
hyaluronic acid are modified to an extent of more than 10%.
30. The method of claim 29, wherein the tissue augmentation is
effective for filling dermal creases or lip reconstruction.
31. A method of sustaining drug release, comprising conjugating one
or more pharmacological compounds to a composition comprising
derivatives of hyaluronic acid comprising disaccharide subunits,
wherein at least one of said disaccharide subunits is a substituted
disaccharide subunit having a substitution at a carboxyl group,
such that the substituted disaccharide subunit is of the formula:
##STR29## wherein each of R' and R'' is a side chain comprising one
or more functional groups selected from the group consisting of
hydrogen; bioactive peptide; alkyl; aryl; alkylaryl; arylalkyl;
substituted alkylaryl containing an atom or atoms of oxygen,
nitrogen, sulfur, or phosphorous; substituted arylalkyl containing
an atom or atoms of oxygen, nitrogen, sulfur, phosphorous, halogen,
or metal ion; and substituted heterocycle containing an atom or
atoms of oxygen, nitrogen, sulfur, phosphorous, halogen or metal
ion; wherein said functional groups within or among each of said R'
or R'' side chains are either bound directly to each other or are
separated by a member selected from the group consisting of ether,
keto, amino, oxycarbonyl, sulfate, sulfoxide, carboxamide, alkyne
and alkene; and wherein each of said R' and R'' side chains
terminates with a terminal functional group selected from the group
consisting of hydrogen, peptide, aldehyde, amine, arylazide,
hydrazide, maleimide, sulfhydryl, active ester, ester, carboxylate,
imidoester, halogen and hydroxyl; and wherein said derivatives of
hyaluronic acid are modified to an extent of more than 10%, wherein
the one or more pharmacological compounds are conjugated to the
derivatives of hyaluronic acid.
32. The method of claim 31, wherein the composition is in a free
form.
33. The method of claim 31, wherein the pharmacological compound is
selected from anti-inflammatories, analgesics, steroids,
cardiovascular agents, anti-tumor agents, immunosuppressants,
sedatives, anti-bacterials, anti-fungals and anti-virals.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] This invention is directed to biomaterials for spatially and
temporally controlled delivery of bioactive agents such as drugs,
growth factors, cytokines or cells. In particular, this invention
teaches versatile methods for chemical crosslinking of high
molecular weight hyaluronic acid under physiological conditions in
situ, to form polymerizable biodegradable materials. The methods
are based on the introduction of functional groups into
hyaluronic-acid (HA) via formation of an active ester at the
carboxylate of the glucuronic acid moiety as an intermediate and
subsequent substitution with a side chain containing a nucleophilic
group on one end and a (protected) functional group on the other
end. The introduced functional groups allow for crosslinking of the
HA derivatives. Crosslinked hyaluronic acid hydrogels of this
invention are useful in various surgical applications and as a
temporary scaffold for tissue regeneration, e.g., in cartilage
repair.
BACKGROUND OF THE INVENTION
Repair of Articular Cartilage
[0002] The failure of regenerating persistent hyaline cartilage by
surgical procedures has prompted investigators to attempt repair
using biological strategies. The biological repair of articular
cartilage is, with a few exceptions, still at an experimental
stage. Biological cartilage repair has been approached in two basic
ways. First, autologous chondrocytes have been transplanted into a
lesion to induce repair (Grande et al., J. Orthop. Res. 7, 208-214
(1989); Brittberg et al., New EngI. J. Med. 331, 889-895 (1994);
Shortkroffet al., Biomaterials 17, 147-154 (1996)). Chondrocytes
may be obtained from a low-loaded area of a joint and proliferated
in culture (see Grande; Brittberg; Shortkroff, supra), or
mesenchymal stem cells may be harvested, e.g., from the iliac crest
marrow, and induced to differentiate along the chondrocyte lineage
using growth factors (Harada et al., Bone 9, 177-183 (1988);
Wakitani et al., J. Bone Joint Surg. 76-A, 579-592 (1994)). The
chondrocyte transplantation procedures currently attempted
clinically, although promising, are hampered because technically
they are very challenging, the cell preparation is very expensive,
and the potential patient pool is limited by age, defect location,
history of disease, etc. Cells have also been transplanted into
cartilage defects in the form of perichondral grafts, e.g.,
obtained from costal cartilage, but with limited success due to the
limit in donor material and the complication of endochondral
ossification of the graft site observed in longterm follow-up
(Aniel et al., Connect. Tissue Res. 18, 27-39 (1988); O'Driscoll et
al., J. Bone Joint Surg. 70-A, 595-606 (1988); Homminga et al.,
Acta Orthop. Scand. 326-329 (1989); Homminga et al., J. Bone Joint
Surg. 72-B, 1003-1007 (1990)). A second approach is aimed at the
recruitment of mesenchymal stem cells from the surrounding
connective tissue, e.g., synovium, using chemotactic and/or
mitogenic factors (Hunziker and Rosenberg, J. Bone Joint Surg.
78-A, 721-733 (1996); see also U.S. Pat. No. 5,368,858). The
availability of growth factors and cytokines in recombinant form
and the lack of complicated cell transplantation make this
procedure a very attractive alternative. The shortcoming of both
procedures is the difficulty to stably anchor the repair-inducing
factors, whether tissue grafts, cells, or growth factors, within
the defect site. Also, outlining of the space that is to be
repaired, e.g., by filling it with a matrix material, appears to be
crucial to recreate a level cartilage surface (Hunziker and
Rosenberg, supra). Thus far, the availability of candidate matrix
materials has been the limiting factor, and anchoring of materials
seeded with chondrocytes and/or chondrogenic factors difficult,
explaining the unsatisfactory results obtained with currently
available materials such as polylactic acid and polyglycolic acid
scaffolds (Freed et al., J. Biomed. Mat. Res. 28, 891-899 (1994);
Chu et al., J. Biomed. Mat. Res. 29, 1147-1154 (1995)); calcium
phosphate minerals (Nakahara et al., Clin. Orthop. 276, 291-298
(1992)), fibrin sealants (Itay et al., Clin. Orthop. 220, 284-303
(1987)), and collagen gels (Wakitani et al., J. Bone Joint Surg.
71-B, 74-80 (1989)). We have developed novel biodegradable
materials based on hyaluronic acid which are optimized for the
biological requirements posed on a repair material in a synovial
joint and which allow in situ polymerization.
Biology of Hyaluronic Acid and Its Therapeutic Use
[0003] Hyaluronic acid (HA) is unique among glycosaminoglycans in
that it is not covalently bound to a polypeptide. HA is also unique
in having a relatively simple structure of repeating nonsulfated
disaccharide units composed of D-glucuronic acid (GIcUA) and
N-acetyl-D-glucosamine (GIcNAc) (Scott et al., The Chemistry.
Biology and Medical Applications of Hyaluronan and Its Derivatives,
T. C. Laurent (ed.), Portland Press, London, (hereinafter
"Hyaluronan and Its Derivatives"), pp. 7-15 (1998)). Its molecular
mass is typically several million Daltons. HA is also referred to
as hyaluronan or hyaluronate, and exists in several salt forms (see
formula I). ##STR1##
[0004] HA is an abundant component of cartilage and plays a key
structural role in the organization of the cartilage extracellular
matrix as an organizing structure for the assembly of aggrecan, the
large cartilage proteoglycan (Laurent and Fraser, FASEB J. 6,
2397-2404 (1992); Morgelin et al., Biophys. Chem. 50, 113-128
(1994)). The noncovalent interactions of aggrecan and link protein
with HA lead to the assembly of a large number of aggrecan
molecules along the HA-chain and mediate retention of aggrecan in
the tissue. The highly negatively charged aggrecan/HA assemblies
are largely responsible for the viscoelastic properties of
cartilage by immobilizing water molecules. A number of cell surface
receptors for HA have been described and shown to play a critical
role in the assembly of the pericellular matrix of chondrocytes and
other cells, e.g., isoforms of CD44 and vertebrate homologues of
Cdc37 (Knudson and Knudson, FASEB J. 7, 1233-1241 (1993);
Grammatikakis et al., J. Biol. Chem. 270, 16198-16205 (1995)), or
to be involved in receptor-mediated endocytosis and degradation of
HA to control HA levels in tissues and body fluids (Laurent and
Fraser, supra; Fraser et al., Hyaluronan and Its Derivatives, pp.
85-92 (1998)). Blocking of the interaction of these receptors with
HA in prechondrogenic micromass cultures from embryonic limb bud
mesoderm inhibits chondrogenesis, indicating that the establishment
and maintainance of a differentiated chondrocyte phenotype is at
least in part dependent on HA and HA-receptor interactions (Maleski
and Knudson, Exp. Cell. Res. 225, 55-66 (1996)).
[0005] HA and its salts are currently being used in therapy for
arthropathies by intraarticular injection (Strachnan et al., Ann.
Rheum. Dis. 49, 949-952 (1990); Adams, Hyaluronan and Its
Derivatives, pp. 243-253 (1998)), in opthalmic surgery for
intraocular lens implantation (Denlinger, Hyaluronan and Its
Derivatives, pp. 235-242 (1998), to promote wound healing in
various tissues (King et al., Surgery 109, 76-84 (1991)), or more
recently, in derivatized and/or crosslinked form to manufacture
thin films which are used for tissue separation (for review see
Laurent and Fraser, supra; Weiss, Hyaluronan and Its Derivatives,
pp. 255-266 (1998); Larsen, Hyaluronan and Its Derivatives, pp.
267-281 (1998); Band, Hyaluronan and Its Derivatives, pp. 33-42
(1998)). Extensive efforts have been made by various laboratories
to produce derivatives of HA with unique properties for specific
biomedical applications. Most of the developments have been
focusing on the production of materials such as films or sponges
for implantation and the substitution of HA with therapeutic agents
for delayed release and/or prolonged effect (for review see Band,
supra; Prestwich et al., Hyaluronan and Its Derivatives, pp. 43-65
(1998); Gustafson, Hyaluronan and Its Derivatives, pp. 291-304
(1998)). Strategies have included esterification of HA (U.S. Pat.
Nos. 4,957,744 and 5,336,767), acrylation of HA (U.S. Pat. No.
5,410,016), and cross-linking of HA using divinyl sulfone (U.S.
Pat. No. 4,582,865) or glycidyl ether (U.S. Pat. No. 4,713,448).
However, the modified HA molecules show altered physical
characteristics such as decreased solubility in water and/or the
chemical reaction strategies used are not designed for crosslinking
of HA under physiological conditions (in an aqueous environment, at
pH 6.5-8.0).
[0006] It is well known that polyaldehydes can be generated by
oxidizing sugars using periodate (Wong, CRC Press, Inc., Boca
Rayton, Fla., pp. 27 (1993); European Patent No. 9615888).
Periodate treatment oxidizes the proximal hydroxyl groups (at C2
and C3 carbons of glucuronic acid moiety) to aldehydes thereby
opening the sugar ring to form a linear chain (Scheme 1). While
periodate oxidation allows for the formation of a large number of
functional groups, the disadvantage is the loss of the native
backbone structure. Consequently, the generated derivative may not
be recognized as HA by cells. In fact, hydrogels formed by using
periodate oxidized HA as a crosslinker, e.g., in combination with
the HA-amines described herein, showed very limited tissue
transformation and poor cellular infiltration in the rat ectopic
bone formation model (FIG. 6). This is in sharp contrast to the
HA-aldehyde derivatives described herein.
[0007] The introduction of free amino groups on HA, which could be
used for further convenient coupling reactions under mild
physiological conditions, has been a subject of great interest.
Previous methods have produced a free amino group on high molecular
weight HA by alkaline N-deacetylation of its glucosamine moiety
(Curvall et al., Carbohydr. Res. 41, 235-239 (1975); Dahl et al.,
Anal. Biochem. 175, 397-407 (1988)). However, concomitant
degradations of HA via beta-elimination in the glucuronic acid
moiety was observed under the harsh reaction conditions needed.
This is of particular concern because low molecular weight HA
fragments, in contrast to high molecular weight HA, have been shown
to be capable of provoking inflammatory responses (Noble et al.,
Hyaluronan and Its Derivatives, pp. 219-225 (1998)). An early
report claimed that carbodilmide-catalyzed reaction of HA with
glycine methyl ester, a monofunctional amine, led to the formation
of an amide linkage (Danishefsky and Siskovic, Carbohydr. Res. 16,
199-201 (1971)). This however, has been proven by a number of
studies not to be the case (Kuo et al., Bioconjugate Chem. 2,
232-241 (1991); Ogamo et al., Carboh dr. Res. 105, 69-85 (1982)).
Under mildly acidic conditions the unstable intermediate
O-acylisourea is readily formed, which in the absence of
nucleophiles, rearranges by a cyclic electronic displacement to a
stable N-acylurea (Kurzer and Douraghi-Zedeh, Chem. Rev. 67,
107-152 (1967)). This O.fwdarw.N migration of the O-acylisourea
also occurs when the nucleophile is a primary amine (Kuo et al.,
supra) and any amide formation that does occur is insignificant as
reported by Ogamo et al., supra. Experiments where high molecular
weight HA (Mr.about.2.times.10 Da) was reacted with an excess of
the fluorescent label 5-aminofluorescine in the presence of the
carbodiimide EDC achieved only 0.86% of theoretical labelling. The
introduction of a terminal hydrazido group on HA with a variable
spacer has recently been achieved and has led to the ability to
conduct further coupling and crosslinking reactions (Pouyani and
Prestwich, Bioconjugate Chem. 5, 339-347 (1994), U.S. Pat. Nos.
5,616,568, 5,652,347, and 5,502,08 1; Vercruysse et al.,
Bioconjupate Chem. 8, 686-694 (1997)).
[0008] It is an objective of this invention to provide a method for
more versatile modification of HA with various functional groups
that allow for crosslinking of the HA derivatives under
physiological conditions. It is another objective that the method
of functionalization does not compromise the molecular weight or
chemical identity (except of the target carboxyl group for
coupling) of HA. It is a further objective that the method of
functionalization provides HA molecules that are well tolerated in
vivo and are biodegradable.
[0009] It is also an objective of this invention to identify HA
derivatives and methodology for in situ polymerization thereof to
provide a biodegradable scaffold for tissue regeneration. It is
another objective that the HA materials can be polymerized in the
presence of cells to serve as a vehicle for cell transplantation.
It is a further objective to provide methodology for
functionalization and cross-linking of HA that allows for
variations in the biomechanical properties of the formed gels as
well as in the sensitivity to cellular infiltration and
degradation.
SUMMARY OF THE INVENTION
[0010] Biomaterials for spatially and temporally controlled
delivery of bioactive agents such as drugs, growth factors,
cytokines or cells, are a key factor for tissue repair. In
particular, in situ polymerizable biodegradable materials are
needed for cartilage resurfacing that are designed to withstand the
mechanical forces in a joint. We have developed a versatile method
for chemical crosslinking of high molecular weight hyaluronic acid
under physiological conditions. The method is based on the
introduction of functional groups into hyaluronic acid by formation
of an active ester at the carboxylate of the glucuronic acid moiety
and subsequent substitution with a side chain containing a
nucleophilic group on one end and a (protected) functional group on
the other end. We have formed hyaluronic acid with amino or
aldehyde functionality, and formed hydrogels with modified
hyaluronic acid and bifunctional crosslinkers or mixtures of
hyaluronic acid carrying different functionalities using active
ester- or aldehyde-mediated reactions. Physical and chemical
properties of the hydrogels of this invention were evaluated using
biomechanical testing, and by assaying sensitivity towards
degradation by glycosidases such as testicular hyaluronidase.
Biocompatibility was evaluated using cell culture assays and
subcutaneous implantation of the hyaluronic acid materials in rats.
This in vivo assay is also the established model for induction of
ectopic bone formation by members of the transforming growth factor
.beta. family (TGF-.beta.), and several crosslinked hyaluronic acid
materials of this invention gave excellent ectopic bone formation
in vivo when loaded with appropriate growth factor(s).
[0011] As set forth below in the detailed description of the
invention, the compositions of the invention have many therapeutic
uses. For example, compositions of the invention may be used to
stem hemorrhage in general surgery, reconstruct nerves and vessels
in reconstructive, neuro- and plastic surgery, and to anchor skin,
vascular, or cartilage transplants or grafts in orthopedic,
vascular, and plastic surgery. Compositions of the invention are
also useful as vehicles for the delivery of cells or bioactive
molecules such as growth factors to stimulate focal repair. Local
delivery of growth factors facilitates wound healing and tissue
regeneration in many situations, not only in promoting bone
formation and stimulating-cartilage repair in orthopedic
procedures, but also, e.g., in treating pathological wound
conditions such as chronic ulcers. These compositions may also
serve as a scaffold to generate artificial tissues through
proliferation of autologous cells in culture. On the other hand,
the anti-adhesive property of some compositions with respect to
cells render such compositions particularly suitable to generate
tissue separations and to prevent adhesions following surgery. The
viscoelastic properties of HA make it particularly well suited for
this purpose, and it is used clinically to achieve temporal pain
relief by repeated intraarticular injections in arthropathies as a
"joint lubricant", as a protective agent for eye irritations and in
ophthalmic surgery, as a barrier to cells in facial and other
reconstructions in plastic surgery and dentistry, in reconstructive
surgery of tendons, in surgical procedures in the urogenital
system, and in thoracic surgery. The injectable nature of the
compositions of the invention also renders them suitable for tissue
augmentation in plastic surgery, where the HA matrix serves
primarily as an inert biocompatible filler material (Balasz and
Laurent, Hyaluronan and Its Derivatives, pp. 325-326 (1998)), e.g.,
for filling dermal creases or lip reconstruction.
[0012] HA hydrogels match several of the desired properties for a
biodegradable material biocompatible with cells. The relatively
simple repetitive structure of HA allows for specific modification
and introduction of a large number of functional groups, for
crosslinking to generate hydrogels with excellent physical
properties. HA hydrogels have also successfully been used as a
delivery vehicle in chondrocyte transplantation studies (Robinson
et al., Calcif. Tissue Int. 46, 246-253 (1990)) and HA has proven
its biocompatibility in various forms in clinical practice (for
review see Laurent and Fraser, supra; Balazs and Laurent,
supra).
[0013] The reaction mechanisms we have explored for in situ
polymerization of HA derivatives are compatible with an aqueous
environment and are non-toxic to cells. The aldehyde-mediated
crosslinking strategies follow reactions occurring physiologically
in crosslinking of fibrillar collagens and elastin. NHS-esters
provide an alternative for rapid formation of stable bonds under
physiological conditions, primarily by reaction with primary
amines. The technology of NHS-ester-mediated protein crosslinking
has been developed for materials with applications in plastic
surgery that require in situ polymerization (U.S. Pat. No.
5,413,791)).
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the results of a ninhydrin test after reductive
alkylation of HA and HA-aldehyde in the presence of putrescine.
Reductive alkylation was carried out with an excess of putrescine
in the presence of pyridine borane. HA or derivatives thereof were
purified by repeated ethanol precipitation prior to detection of
free amino groups on the HA chain by using the ninhydrin test
(Sheng et al., Anal. Biochem. 211, 242-249 (1993)).
[0015] FIG. 2 shows .sup.1H NMR of native HA (FIG. 2A) and an
HA-derivative with protected aldehyde functionality (FIG. 2B) in
D.sub.2O at 300 Mhz. Peaks are assigned as indicated on the
structural formula.
[0016] FIG. 3 shows .sup.1H NMR of HA-derivatives with amine
functionality formed from putrescine (FIG. 3A), histidine (FIG.
3B), lysine (FIG. 3C), and adipic dihydrazide (FIG. 3D) in D.sub.1O
at 300 Mhz. Peaks are assigned as indicated on the structural
formula.
[0017] FIG. 4 shows digestion of crosslinked HA hydrogels with
hyaluronidase. In FIG. 4A, HA-hydrogels were formed by crosslinking
12 mg/ml highly modified (.about.65-70%) HA-amine (adipic
dihydrazido-HA) with 15 mg/ml (SPA).sub.2-PEG. Gels were incubated
with different concentrations of bovine testicular hyaluronidase
for the indicated time and the degradation of the gels was measured
by the release of glucuronic acid into the supernatant using the
carbazole method (Bitter and Muir, Anal Biochem. 4, 330-334
(1962)). In FIG. 4B, HA-hydrogels were formed by crosslinking 12
mg/ml optimally modified (.about.20-25%) HA-amine (adipic
dihydrazido-HA) with 15 mg/ml (SPA).sub.2-PEG (.diamond.); 12 mg/ml
highly modified (.about.65-70%) adipic dihydrazido-HA with 15 mg/ml
(SPA).sub.2-PEG (.DELTA.); 12 mg/ml optimally modified
(.about.20-25%) lysine methylester-HA with either 15 mg/ml
(SPA).sub.2-PEG (.LAMBDA.) or 0.44 mg/ml glutaraldehyde
(.quadrature.), and 12 mg/ml optimally modified (.about.10-15%)
diaminobutyl-HA with 15 mg/ml (SPA).sub.2-PEG (o). Gels were
incubated with different concentrations of bovine testicular
hyaluronidase for the indicated time and the degradation of the
gels was measured as in FIG. 4A above.
[0018] FIG. 5 shows phase contrast images of cells cultured on
different crosslinked HA hydrogels. FIG. 5A: Dedifferentiated
chondrocytes cultured on a hydrogel formed from highly modified
(.about.65-70%) HA-amine (adipic dihydrazido-HA) crosslinked with 5
mg/ml (SPA).sub.2-PEG aggregate as a consequence of inability to
adhere to substratum. FIG. 5B: Cells cultured on a hydrogel made up
by the same HA-amine crosslinked with 0.25 mg/ml glutaraldehyde
show a rounded morphology and no aggregation indicating that they
are able to adhere to the substratum. FIG. 5C: Cells cultured on a
hydrogel formed from the HA-amine (adipic dihydrazido-HA) modified
to a degree of .about.20-25% and crosslinked with 2 mg/ml
(SPA).sub.2-PEG adhere to the substratum, spread and subsequently
infiltrate the hydrogel. All images show cells 24 h post seeding
but morphology remains the same even after several days in
culture.
[0019] FIG. 6 shows in vivo evaluation of HA hydrogels formed from
different HA derivatives using aldehyde-mediated crosslinking.
Subcutaneous implantation in rats of HA hydrogels consisting of
(FIG. 6A) 12 mg/mn optimally modified (.about.20-25%) HA-amine
(adipic dihydrazido-HA) crosslinked with 0.25 mg/ml glutaraldehyde,
(FIG. 6B) 7 mg/ml of the same HA-amine crosslinked with 7 mg/ml
HA-aldehyde (periodate oxidized), (FIG. 6C) 7 mg/ml of the same
HA-amine crosslinked with 7 mg/ml HA-aldehyde (deprotected
amino-dimethyl acetal-HA, .about.10-15% modified), or (FIG. 6D) 7
mg/ml of the same HA-amine crosslinked with 7 mg/ml HA-aldehyde
(deprotected hydrazido-dimethyl acetal-HA, .about.40-45% modified).
The hydrogels also contained 1 mg/ml prefibrillized intact collagen
type I, 200 .mu.g/ml BMP-2 and 500 ng/ml IGF-1 to induce bone
formation. Tissue specimens were harvested 10 days post
implantation, fixed in formalin and processed for histology by
paraffin embedding. Sections were stained with Haematoxylin/Eosin.
mn, matrix material (note: matrix material shrinks during
dehydration); s, skin (indicates orientation of implant).
[0020] FIG. 7 shows in vivo evaluation of HA hydrogels crosslinked
with different NHS-esters. Subcutaneous implantation in rats of HA
hydrogels consisting of (FIG. 7A) 12 mg/ml highly modified
(.about.65-70%) HA-amine (adipic dihydrazido-HA) crosslinked with
15 mg/nl (SPA).sub.2-PEG, (FIG. 7B) 12 mg/ml optimally modified
(.about.20-25%) HA-amine (adipic dihydrazido-HA) crosslinked with
15 mg/ml SPA.sub.2-PEG, or (FIG. 7C) 12 mg/ml of the same optimally
modified F-Lamine crosslinked with 3 mg/ml DTSSP (crosslinker
concentrations are equal on a molar basis). The hydrogels also
contained 1 mg/ml prefibrillized intact collagen type I, 200
.mu.g/ml BMP-2 and 50 ng/ml TGF-.beta.2 to induce bone formation.
Tissue specimens were harvested 10 days post implantation, fixed in
formalin and processed for histology by paraffin embedding.
Sections were stained with Haematoxylin/Eosin. m, matrix material
(note: matrix material shrinks during dehydration); s, skin
(indicates orientation of implant).
[0021] FIG. 8 shows differential effect of growth factors
incorporated into HA hydrogels on tissue transformation.
Subcutaneous implantation in rats of the HA hydrogel formed from 12
mg/ml optimally modified (.about.20-25%) HA-amine (adipic
dihydrazido-HA) crosslinked with 15 mg/ml (SPA).sub.1-PEG. The
hydrogels also contained 1 mg/ml prefibrillized intact collagen
type I, and were supplemented either with 200 .mu.g/ml BMP-2 and
500 ng/ml IGF-1 (FIG. 8A), or 200 .mu.g/ml BMP-2 and 50 ng/ml
TGF-.beta.2 (FIG. 8B). Tissue specimens were harvested 10 days post
implantation, fixed in formalin and processed for histology by
paraffin embedding. Sections were stained with
Haematoxylin/Eosin.
BRIEF DESCRIPTION OF THE REACTION SCHEMES
[0022] Scheme 1 illustrates periodate oxidation of hyaluronic
acid.
[0023] Scheme 2 illustrates coupling of amines to hyaluronic acid
with EDC via an active triazole ester intermediate.
[0024] Scheme 3 illustrates coupling of amines to hyaluronic acid
with EDC via an active N-hydroxysuccinimde ester intermediate.
[0025] Scheme 4 illustrates crosslinking of amnine functionalized
hyaluronic acid with various bifunctional N-hydroxysuccinimde ester
crosslinkers to form hydrogels. (1. (SPA).sub.2-PEG; 2. DTSSP).
[0026] Scheme 5 illustrates crosslinking of amine functionalized
hyaluronic acid with glutaraldehyde to form hydrogels. In addition
to the conventional reaction of aldehydes with amines that results
in the formation of a Schiff base, glutaraldehyde is also known to
undergo polymerization by aldol condensation yielding polymers with
.alpha.,.beta.-unsaturated aldehydes at neutral or slightly
alkaline pH (Richards and Knowles, J. Mol. Biol. 37, 231-233
(1968)). Subsequent, nucleophilic addition of amines at the
ethylenyl double bond creates a stable crosslink.
[0027] Scheme 6 illustrates formation of hydrogels with aldehyde
functionalized hyaluronic acid. (1. amine functionalized HA; 2.
bifunctional amine) ##STR2## ##STR3## ##STR4## ##STR5## ##STR6##
##STR7##
DETAILED DESCRIPTION OF THE INVENTION
[0028] Using the methods of our invention, we generate an activated
form of HA that differs minimally from native HA to conserve its
unique physico-chemical properties. We also effect a minimal change
affecting only a relatively small number of dissaccharide units of
native HA so that we do not alter its property to serve as a cell
substratum.
[0029] We initially attempted to generate an aldehyde derivative of
HA by reduction of the carboxyl groups of the glucuronic acid
moieties into aldehydes using 9-borabicyclo-3,3-nonane, a method
that allows direct conversion of the carboxylic acid into the
aldehyde (Cha et al., Bull. Korean Chem. Soc. 9, 48-52 (1988), Cha
et al., Org. Prep. Proc. Int. 21, 451-477 (1989)): HA--COOH
(I).fwdarw.HA--CHO (II)
[0030] However, even though preliminary testing indicated the
conversion of the carboxyl groups into aldehydes to a degree of
approximately 5-10% (FIG. 1), mixtures of concentrated, viscous
HA-aldehyde solutions (.about.10 mg/ml) with `small` polyarnines
such as putrescine, lysine, polylysine, histidine, or polyhistidine
did not generate stable gels in a reasonable time frame to be
suitable for in situ polymerization. It is important to note that
the chemical properties of HA are determined not merely by its
functional groups per se but also by the accessibility of these
functional groups of HA in an aqueous environment, which is related
to the overall conformational structure and rheological properties
of HA. HA behaves like a hydrogel in an aqueous media even in the
absence of crosslinks because it forms a network stabilized by
hydrogen bonds and van der Waals forces (Laurent and Fraser,
supra). To increase the accessibility of functional groups, we
introduced a spacer between the functional group and the HA
chain.
Introducing a Functionalized Side Chain Onto HA
[0031] We subsequently developed methodology for introducing side
chains into HA by carbodimide-mediated coupling of primary or
secondary amines to the carboxyl group of the glucuronic acid
moiety using an active ester intermediate. We have used this
methodology to generate HA with different terminal functional
groups for crosslinking including acetals, aldehydes, amines, and
hydrazides. A wide range of functionalized amines are commercially
available which allows us to introduce a wide variety of different
functional groups useful for crosslinking under physiological
conditions using this methodology, including maleimides that react
specifically with sulfflydryls or arylazides for photocrosslinking
besides the amines and aldehydes described below.
[0032] Direct carbodiimide-mediated coupling of amines to the
carboxyl group of HA in an aqueous environment, e.g., with EDC
(1-ethyl-3-[3-dimethylarninopropyl]carbodiimide), does not yield
the predicted product since the O-acyl isourea that is formed as a
reactive intermediate rearranges rapidly to a stable N-acyl urea
(Kuo et al., supra). We have demonstrated that by "rescuing" the
active O-acyl isourea by formation of a more hydrolysis resistant
and non-rearrangable active ester intermediate, the coupling of
primary amines to HA is possible. A wide variety of active
carboxylic esters exist and could be formed for further reaction
including NHS-esters, nitrophenol esters, triazole esters, sulfonic
esters, etc., as long as the reagent for their preparation is
soluble in H.sub.2O or in other polar solvents such as
dimethylsulfoxide or dimethylformamide. HA is soluble in H.sub.2O
or other aprotic polar solvents in native form and when prepared as
a sodium salt or when prepared as a tetrabutylammonium salt as
described in U.S. Pat. No. 4,957,744, respectively. We have formed
active esters of HA with 1-hydroxybenzotriazole (HOBT) or
N-hydroxysulfo-succinimide using the H.sub.2O soluble carbodiimide
EDC for coupling. Nucleophilic addition to the ester formed from
HOBT requires the amine to be presented in unprotonated form at
acidic pH (about 5.5 to 7.0). Only a limited number of amines
including hydrazines and activated amines, e.g., ethylene diamine,
have pKa values in a suitable range and are consequently
unprotonated and reactive with the ester-intermediate formed with
HOBT (Scheme 2). Simple primary amines, e.g., putrescine, which
typically have pKa values>9 do not form significant amounts of
adduct under acidic coupling conditions. The
N-hydroxysulfosuccinimide-derived intermediate allows for the
coupling reaction to be carried out at neutral pH (about 7.0 to
8.5) and consequently yields products by reaction with simple
primary amines (Scheme 3).
[0033] Consequently, this methodology allows for the following
reactions to be carried out: HA--COOH (I)+H.sub.2N--R
(III).fwdarw.HA--CO--NH--R (IV) HA--COOH (I)+R'--NH--R
(V).fwdarw.HA--CO--NR'--R (VI) wherein R and R' are alkyl, aryl,
alkylaryl or arylalkyl side chains which may contain hetero atoms
such as oxygen, nitrogen, and sulfur. The side chain may be
branched or unbranched, and be saturated or may contain one or more
multiple bonds. The carbon atoms of the side chain may be
continuous or may be separated by one or more functional groups
such as an oxygen atom, a keto group, an amino group, an
oxycarbonyl group, etc. The side chain may be substituted with aryl
moieties or halogen atoms, or may in whole or in part be formed by
ring structures such as cyclopentyl, cyclohexyl, cycloheptyl, etc.
The side chain may have a terminal functional group for
crosslinking such as aldehyde, amine, arylazide, hydrazide,
maleimide, sulfhydryl, etc. The side chain may be a bioactive
peptide, e.g., containing receptor binding sites, crosslinking
sites for transglutarninases, or proteolytic cleavage sites.
[0034] Terminal functional groups of the side chain useful for
crosslinking of HA under physiological conditions may be selected
from the following list:
[0035] 1. Aldehydes, see Examples H.sub.2N--R--CHO (VII)
[0036] 2. Amines, see Examples H.sub.2N--R--NH.sub.1 (VIII)
[0037] 3. Arylazides, e.g., 4-(p-azidosalicylamido)butylamine
##STR8##
[0038] 4. Maleimides, e.g.,
4-(N-maleimidomethyl)cyclohexane-1-carboxylhydrazide ##STR9##
[0039] 5. sulfhydryls, e.g., S-methylsulfide cysteine
H.sub.2N--R--SH (XI)
[0040] 6. Peptides, e.g., H.sub.2N-APQQEA, comprising substrate
sites for enzymatic crosslinking, e.g., by transglutarninases
(Parameswaran et al., Proc. Natl. Acad. Sci. U.S.A. 87, 8472-8475
(1990); Hohenadl et al., J. Biol. Chem. 270, 23415-23420
(1995)).
[0041] The carbodiimides useful in this reaction may be represented
by the following formula: R--N.dbd.C.dbd.N--R' (XII) wherein R and
R' comprise side chains of variable structure as described above in
detail. Carbodiimides which are soluble in an aqueous media are
preferred.
[0042] The active ester may be of the following class and be formed
by carbodiimide-mediated coupling of a compound for preparation of
these active esters known to a person in the art:
[0043] 1. Triazole Esters, e.g. 1-hydroxybenzotriazole
##STR10##
[0044] 2. NHS-Esters, e.g. N-hydroxysulfosuccinimide ##STR11##
[0045] 3. nitrophenol esters, e.g. p-nitrophenol ##STR12##
Preparation of HA-aldehyde Derivatives
[0046] A side chain containing a protected aldehyde in the form of
an acetal was prepared as follows.
N-(2,2-dimethoxyethyl)-4-(methoxycarbonyl)butanamide was obtained
from aminoacet-aldehyde dimethyl acetal and mono-methyl succinate
using EDC coupling. An amino group for the coupling to HA was
subsequently introduced by reacting the product with hydrazine,
yielding the desired side chain with the protected aldehyde,
N-(2,2-dimethoxyethyl)-4-(hydrazido)butanamide. The side chain was
coupled to HA using HOBT and EDC (Scheme 2). An acetal side chain
with a simple primary amine, 1-aminoethyl-dimethylacetal, was
conjugated to HA using N-hydroxysuccinimide and EDC (Scheme 3). The
HA-derivatives were purified by ethanol precipitation. The nature
of the HA-derivatives was confirmed by .sup.1H NMR (FIG. 2). The
HA-acetal derivatives are easily activated to the reactive
aldehydes by mild acid treatment. Other HA-aldehyde derivatives
with variations in the length of the side chain have been prepared
in a similar manner. See Examples 1-3.
Preparation of HA-amine Derivatives
[0047] Diaminoethane, lysine methyl ester, histidine, and adipic,
succinic or suberic dihydrazide was coupled to HA using HOBT and
EDC (up to 5-fold excess depending on the desired degree of
modification) and adjusting the pH to .about.6.5 by repeated
addition of 0.1M HCl during the reaction (Scheme 2). HA-derivatives
were also prepared in a similar manner using
N-hydroxysulfosuccinimide and primary amines containing
unconjugated amino groups with a higher pKa (>9) such as
1,4-diaminobutane or 1,6 diaminohexane (Scheme 3). The HA
derivatives were purified by repeated ethanol precipitation and by
extensive dialysis, and the nature of the HA derivatives was
confirmed by .sup.1H NMR (FIG. 3). The degree of modification was
calculated from the NMR data and found to be as high as 70%.
Reaction conditions were subsequently adjusted such that a degree
of modification of approximately 20% was achieved. Limiting the
amount of carbodiimide proved to be most successful in controlling
the degree of modification. A degree of modification of 10-25%
yielded efficient crosslinking but also a molecule that would still
be recognized by glycosidases and by HA receptors as HA and thus
allow for recognition and processing of the material by cells (see
below). Similar HA derivatives were synthesized using succinic,
adipic or suberic dihydrazide or diaminoethane, -butane, or -hexane
to study the effect of the length of the spacer separating the
introduced functional group from the HA-chain on the subsequent
crosslinking. See Examples 4-8.
Crosslinked HA Hydrogels
[0048] The functionalized HA molecules can be crosslinked by
reacting HA derivatives with different functionalities or using
homo- or heterobifunctional crosslinkers which are available in
large variety. The following basic reaction schemes are suitable
for crosslinking of the described forms of modified HA (see
Examples 9-12):
[0049] 1. Aldehyde-Mediated Crosslinking
R.sub.1--CHO+H.sub.2N--R.sub.2.fwdarw.R.sub.1--CH.dbd.N--R.sub.2
[0050] 2. Active Ester-Mediated Crosslinking ##STR13##
[0051] 3. Maleimide-Mediated Crosslinking ##STR14##
[0052] 4. Photo-Crosslinking ##STR15##
[0053] 5. Enzymatic Crosslinking (transglutaminase)
R.sub.1--(CH).sub.2--CONH.sub.2+H.sub.2N--R.sub.2.fwdarw.R.sub.1--(CH).su-
b.2--CO--NH--R.sub.2
[0054] Crosslinking of the HA-amine derivatives (Mr.about.10.sup.6)
with bifunctional active esters, e.g.
polyethyleneglycol-bis-succinimnidyl-propionate [(SPA).sub.2-PEG]
and reducible 3,3'-dithiobis(sulfo-succinimidyl-propionate) (DTSSP)
(Scheme 4), or bifunctional aldehydes, e.g. glutaraldehyde (Scheme
5), generated excellent hydrogels. Stable gels could be formed by
crosslinking 5 to 25 mg/ml HA derivative with >0.05 mM aldehyde
or >0.2 mM active ester (numbers are reflecting functional group
concentrations). Optimal gels were generated by crosslinking
10-15mg/ml HA derivative, modified to a degree of about 10-25%,
with about 0.2 mM aldehyde or 0.6 mM active ester. Similarly,
crosslinking of the HA-aldehyde derivatives (Mr.about.10.sup.6)
(optimally about 10-15 mg/ml) with bifunctional amines (optimally
about 0.2 mM) yielded excellent gels (Scheme 6). Conjugated amines
such as dihydrazines or benzylamines are required for in situ
polymerization of HA in this case to resonance stabilize the
instable Schiff base product formed by reaction of an aldehyde with
a primary amine (i.e. hydrazines yield a more stable hydrazone
linkage). Hydrogels were also formed from an equimolar mixture of
HA-aldehyde derivatives and the different HA-amine derivatives
(Scheme 6). Optimal gels were formed with .about.15 mg/ml of the HA
derivatives. At the optimal concentrations of HA and crosslinker,
gelation occurred typically in about 30 sec. to 5 min. which is
suitable for in situ polymerization. The crosslinked HA hydrogels
were sensitive to glycosidases, i.e. testicular hyaluronidase,
indicating that they are biodegradable (FIG. 4).
[0055] A number of different tests including cell culture assays
and animal experiments served to assess biocompatibilty of the
formulated biomaterials. Embedding of chondrocytes into the
polymerizing HA hydrogels showed that neither aldehyde nor
NHS-ester-mediated crosslinking was toxic to cells at the
concentrations employed. Seeding of cells on top of prepolymerized
HA hydrogels showed a wide variety of cellular behaviours depending
on the nature of the crosslinker and crosslinking density (FIG. 5).
Highly crosslinked HA hydrogels were impenetrable to cells (FIGS.
5, A and B), while optimally crosslinked gels were infiltrated
(FIG. 5C). Supplementation of the HA hydrogels with cell adhesion
molecules such as fibronectin (in the range of 0.1 to 1 mg/ml) did
induce adhesion and spreading of cells on the materials independent
of the nature of the crosslinker and the crosslinking density, but
did not change the results with regard to cell infiltration,
demonstrating that the lack of infiltration is due to the high
crosslinking density and not the absence of cell-matrix
interactions. See below and FIG. 7.
[0056] Subcutaneous implantation of biomaterials in rats is the
established model for evaluation of biocompatibility of
biomaterials (Laurencin et al., J. Biomed. Mat. Res. 24, 1463-1481
(1990)) and for induction of ectopic bone formation by members of
the TGF-.beta. gene family, and bone morphogenetic proteins (BMP)
in particular (Wang et al., Proc. Natl. Acad. Sci. U.S.A. 87,
2220-2224 (1990); Sampath et al., J. Biol. Chem. 267, 20352-20362
(1992)). Taking into consideration the cell culture results, we
have formulated a number of HA hydrogels for in vivo
biocompatibility testing in this model. Implantation of
prepolymerized HA hydrogel discs loaded with recombinant BMP-2 and
IGF-1 or TGF-.beta.2 subcutaneously in rats showed a mild fibrosis
with a varying degree of cartilage and bone formation depending on
the nature of the HA biomaterial (FIGS. 6 and 7). The growth
factors were mixed with the HA derivatives prior to gelling and the
induction of bone formation suggests that neither reaction
mechanism used for HA crosslinking (aldehyde or active
ester-mediated reactions) significantly affected the biological
activity of the growth factors. Little inflammation was observed
with active ester crosslinked HA-amine derivatives (FIG. 7) or with
HA-amine derivatives crosslinked with various HA-aldehyde
derivatives (FIGS. 6B-6D) while a stimulation of foreign body giant
cells was seen when the same HA-amine derivatives were crosslinked
with glutaraldehyde (FIG. 6A). The degree of modification of HA
strongly affected the resorption and transformation rate of the
biomaterials (FIGS. 7A, 7B). Nevertheless, limited bone formation
was seen even with a biomaterial formed from a highly modified
(65-70%) HA-amine derivative (FIG. 7A). The absence of bone
formation with a smaller bifunctional NHS-ester crosslinker
indicates that the size of the generated crossbridge is crucial for
resorption and cellular infiltration (FIG. 7C). This is probably
due to the difference in pore size of the material formed with
crosslinkers of different sizes. The infiltration and
transformation rate was similar with BMP-2/IGF-1 and
BMP-2/TGF-.beta.2 loaded biomaterials, indicating that the
resorption rate is a material property. However, at ten days
post-implantation, the newly formed tissue was largely cartilage in
the first group and largely bone in the second group (FIG. 8),
exemplifying the angiogenic effect of TGF-.beta.2 (Yang and Moses,
J. Cell. Biol., 111, 731-741 (1990)). This demonstrates that the
biological activity of the HA material can be modulated by
inclusion of different bioactive factors. The lack of significant
adverse effects and the demonstration of the desired biological
activity of these novel HA biomaterials in vivo demonstrates their
usefulness as a delivery vehicle for cells and growth factors in
the field of tissue regeneration.
[0057] There are several approaches to the production of HA,
including extraction from tissue and biosynthesis. Extraction from
tissue typically uses fresh or frozen cocks' combs (U.S. Pat. No.
5,336,767), although other tissues including the synovial fluid of
joints (Kvam et al., Anal. Biochem. 211, 44-49 (1993)), human
umbilical cord tissue, bovine vitreous humor, and bovine tracheae,
have been used. It is also possible to prepare HA by
microbiological methods, such as by cultivating a microorganism
belonging to the genus Streptococcus which is anhemolytic and
capable of producing HA in a culture medium (U.S. Pat. Nos.
4,897,349; 4,801,539; 4,780,414; 4,517,295; 5,316,926). The HA raw
material for preparing the compositions of the invention preferably
consists of high molecular weight HA, more preferably of molecular
weight greater than 0.5 million daltons, and more preferably of
molecular weight greater than one million daltons. The HA raw
material for the compositions of examples of this invention
described herein was obtained from Genzyme Corp. (Cambridge,
Mass.), and had a molecular weight greater than one million
daltons. The size of the HA was unchanged after derivatization.
[0058] The compositions of the invention have many therapeutic
uses. The fact that the compositions may be cured in a surgically
practical time frame of one to five minutes in situ with concurrent
crosslinking to the tissue surfaces allows for employment as a
tissue glue. Many situations in various surgical applications
require such adhesives. For example, the compositions of the
invention may be used to stem hemorrhage in general surgery,
reconstruct nerves and vessels in reconstructive, neuro- and
plastic surgery, and to anchor skin, vascular, or cartilage
transplants or grafts in orthopedic, vascular, and plastic surgery.
Those of skill in the art may choose and design particular
embodiments of the invention which are particularly suitable for a
desired application, by adjusting several factors, including: (1)
the degree of functionalization of HA, which affects the
crosslinking density of the material and interaction with cellular
proteins, including receptors and glycosidases; (2) the
concentration of the crosslinker, which affects the crosslinking
density of the material; (3) the size of the generated
cross-bridge, which affects the pore size of the material; (4) the
nature of the crosslinking mechanism, which determines
polymerization time and the specificity of the reaction; and (5)
the nature of the cross-bridge, which provides biological cues. See
FIGS. 4, 5, and 7 for data concerning HA hydrogels with different
crosslinking densities and pore sizes. Generally, active ester- or
photo-crosslinking are preferred to form materials for applications
requiring fast gelation and strong bonding with tissue surfaces,
such as tissue glues. Materials with anti-adhesive properties,
which are useful to form tissue separations or for tissue
augmentation, are formed from highly modified HA derivatives with
low molecular weight crosslinkers, which generates a dense material
with very small pores, thereby minimizing cell adhesion and
infiltration. Conversely, biodegradable scaffolds for tissue repair
are formed from HA with a limited degree of derivativization and
high molecular weight crosslinkers, which generate a porous,
biodegradable material. The crossbridge may even contain biological
cues, such as peptide sequences, which facilitate material
degradation by, for example, proteolysis or cellular infiltration
(e.g., the RGD sequence).
[0059] Compositions of this invention were designed to serve as a
vehicle for the delivery of cells or bioactive molecules such as
growth factors to stimulate focal repair. The crosslinked HA
derivatives are porous hydrogels in which biologically or
therapeutically active compounds (e.g., growth factors, cytokines,
drugs, and the like) can be physically or chemically incorporated.
These compounds will then be subject to sustained release by
chemical, enzymatic, and physical erosion of the hydrogel and/or
the covalent linkage between the HA chain and biologically active
compound over a period of time. Local delivery of growth factors
with such a scaffold facilitates wound healing and tissue
regeneration in many situations. For example, the compositions of
the invention may be used not only to promote bone formation and
stimulate cartilage repair in orthopedic procedures, as described
more fully below, but also to treat pathological wound conditions
such as chronic ulcers. They may also serve as a scaffold to
generate artificial tissues, e.g., cartilage (Hauselmann et al.,
Am. J. Physiol. 271, C742-752 (1996)), through proliferation of
autologous cells in culture. Similar procedures for generation of
equivalents of other tissues or organs, including skin, liver, and
others, in culture may be developed in the future and may be used
in combination with the compositions of the invention.
[0060] Highly crosslinked materials have an anti-adhesive property
with respect to cells, and such compositions may be used to
generate tissue separations and to prevent adhesions following
surgery. See FIGS. 5A and 7C, showing highly modified HA-amine,
i.e., adipic dihyrazido HA, preferably crosslinked with low
molecular weight bifunctional NHS-ester. The viscoelastic
properties of HA make it particularly well suited for this purpose,
and it is used clinically to achieve temporal pain relief by
repeated intraarticular injections in arthropathies as a "joint
lubricant", and as a protective agent for eye irritations and in
ophthalmic surgery. The technique of tissue separation is used in
facial reconstruction in plastic surgery and dentistry. Prevention
of the formation of adhesions is particularly relevant in
reconstructive surgery of tendons, in surgical procedures in the
urogenital system, and in thoracic surgery. Many different HA-based
materials are already in clinical use in these areas. (See products
manufactured by Anika Therapeutics, Inc. (Woburn, Mass.),
Biomatrix, Inc. (Ridgefield, N.J.), Genzyme Corp. (Cambridge,
Mass.), and Fidia, S.p.A. (Abano Terme, Italy)). Those of skill in
the art may choose and design particular embodiments of the
invention which are particularly suitable for a desired application
by selecting distinct features as outlined above.
[0061] The injectable nature of the compositions of the invention
also renders them suitable for tissue augmentation in plastic
surgery, where the HA matrix serves primarily as a biocompatible
filler material, e.g., for filling dermal creases or lip
reconstruction. Again, those of skill in the art may choose and
design particular embodiments of the invention which are
particularly suitable for a desired application, as outlined
above.
[0062] The half-life of pharmacological compounds, both synthetic
and biological, has been shown to be drastically increased when
delivered in a form conjugated to HA (Larsen and Balazs, Adv. Drug
Delivery Rev. 7, 279-293 (1991), Drobnik, J., Drug Delivery Rev. 7,
295-308 (1991)). The functionalized forms of HA provided by this
invention allow for easy substitution with pharmacologically active
agents, such as anti-inflammatories, analgesics, steroids,
cardiovascular agents, anti-tumor agents, immunosuppressants,
sedatives, anti-bacterial, anti-fungal, and anti-viral agents,
etc., and may be used for sustained drug release over time, either
locally in hydrogel form or systemically in free form.
[0063] In orthopedic surgery, the functionalized forms of HA of
this invention have applications as a tissue glue or bioactive
matrix material in the treatment of chondral and osteochondral
fractures, osteochondritis dissecans, meniscal tears, as well as
ruptured ligaments, tendons, or myotendineous junctions. The HA
materials of this invention may serve to facilitate anchorage of
chondral or osteochondral transplants or grafts, or other
biological or artificial implant materials, or to stimulate new
bone or cartilage formation by serving as a scaffold for cells or
as a delivery vehicle for growth factors. One general approach to
promote articular cartilage repair based on the compositions of the
invention comprises using: (1) in situ polymerized HA hydrogel as a
matrix to fill the defect which is to be repaired and to provide a
scaffold for repair cells, (2) an optional chemotactic agent to
attract repair cells to the matrix and defect site, or
alternatively, autologous chondrocytes or mesenchymal stem cells,
(3) an optional factor to promote cellular proliferation of repair
cells in the matrix and defect site; (4) sustained release of a
transforming factor by the HA hydrogel over time to promote
differentiation of the repair cells into chondrocytes which produce
new cartilage; and (5) an optional anti-angiogenic factor to
prevent vascularization and endochondral ossification of the newly
formed cartilage. Examples of suitable factors are known to those
skilled in the art, and may be found in, e.g., U.S. Pat. No.
5,368,858.
[0064] Delivery of growth factors in active form may require
supplementation of the HA hydrogels with additional ingredients,
such as growth factor binding molecules like heparin and collagen.
For example, for cartilage repair, crosslinked hyaluronic acid
hydrogels that are rapidly infiltrated by cells such as those
formed from an HA-amine derivative crosslinked with a polyvalent
high molecular weight NHS-ester crosslinker, e.g., (SPAs-PEG, are
selected which are resorbed and replaced by repair tissue within
about 2 to 3 weeks. In some cases, cells and/or growth factors may
be mixed in prior to gelling.
[0065] The following are illustrative examples, which are not
intended to limit the scope of the present invention.
EXAMPLES
Example 1
[0066] Preparation of
N-(2,2-dimethoxyethyl)-4-(methoxycarbonyl)butanamide (1) - EDC
(4.98 g, 0.026 mol) was added to a mixture of aminoacetaldehyde
dimethyl acetal (2.18 ml, 20 mmol) and methyl monoester of succinic
acid (2.64 g, 20 mmol) in 75 ml of dichloromethane, and the
reaction mixture stirred for 24 h at room temperature. The solution
was extracted successively with 50 ml each of ice cold solutions of
0.75M sulfuric acid, 1M NaCl, saturated sodium bicarbonate, and 1M
NaCl. The organic phase was collected and dried with sodium
sulfate. The solvent was evaporated under reduced pressure yielding
a syrup, which showed a single spot on charring upon TLC in solvent
A (R.sub.f 0.75) and solvent B (R.sub.f 0.24). The apparent yield
of 1 was 65%.
[0067] .sup.1H NMR in CDCl.sub.3 .delta. 5.70 (bs, 1H, NH), 4.34
[t, 1H, CH--(OCH.sub.3), 3.67 (s, 3H, COOCH.sub.3), 3.43-3.35 (s
and t, 8H, CH.sub.3OC and CHCH.sub.2NH), 2.38-2.26 (m, 4H,
CH.sub.2CO).
[0068] Formation of Acyl-hydrazide (2) from 1-Anhydrous hydrazine
(248 .mu.l, 7.9 mmol) was added to a solution of 1 (1.73 g, 7.9
mmol) in 5 ml of anhydrous methanol. The mixture was stirred at
room temperature overnight and the solvent subsequently evaporated
under reduced pressure yielding a solid residue. The residue was
dissolved in H.sub.2O (6 ml) and extracted three times with an
equal volume of dichloromethane. The aqueous solution was
evaporated to dryness under reduced pressure and then further dried
overnight in vacuo. The crystaline solid (1.04 g, 82% yield) was
homogeneous on TLC in solvent A (R.sub.f 0.10) when visualized by
charring. The .sup.1H NMR spectrum indicated the loss of the ester
methoxy group when compared to 1.
[0069] Preparation of Hydrazido-dimethyl acetal-HA (formula XIX)
-Sodium hyaluronate (100 mg, 0.25 mmol) and
N-(2,2-dimethoxyethyl)-4-(hydrazido)butanamide (2) (1.646 g, 7.5
mmol) was dissolved in H.sub.2O (40 ml, 2.5 mg/ml HA). The pH was
adjusted to 6.5 and HOBT (169 mg, 1.25 mmol) predissolved in a 1:1
mixture of water and DMSO (1 ml) and EDC (240 mg, 1.25 mmol) was
added and the reaction mixture was stirred overnight. The pH was
subsequently adjusted to 7.0 with 1M NaOH and NaCl added to produce
a 5% w/v solution. HA was precipitated by addition of three volume
equivalents of ethanol. The precipitate was redissolved in H.sub.2O
at a concentration of approximately 5 mg/ml and the precipitation
repeated twice. The purified product was freeze dried and kept at
4.degree. C. under N.sub.2. See FIG. 2B for NMR data of the
product. ##STR16##
Example 2
[0070] Preparation of Aminoacetaldehyde-dimethyl acetal-HA (formula
XX)-Sodium hyaluronate (100 mg, 0.25 mmol) and
2,2-dimethoxyethylarnine (0.788 g, 7.5 mmol) was dissolved in
H.sub.2O (40 ml, 2.5 mg/ml HA). The pH was adjusted to 7.5 and
NHS.SO.sub.3Na (268 mg, 1.25 mmol) and EDC (240 mg, 1.25 mmol) was
added and the reaction mixture was stirred overnight. The pH was
subsequently adjusted to 7.0 with 1M NaOH and NaCl added to produce
a 5% w/v solution. HA was precipitated by addition of three volume
equivalents of ethanol. The precipitate was redissolved in H.sub.2O
at a concentration of approximately 5 mg/ml and the precipitation
repeated twice. The purified product was freeze dried and kept at
4.degree. C. under N.sub.2. ##STR17##
Example 3
[0071] Deprotection of HA-acetals to form HA-aldehydes--The acetal
modified HA(formula XII) was dissolved in H.sub.2O to a
concentration of 5-10 mg/ml and 1M HCl was added to give a final
concentration of 0.025M. The solution was then allowed to stand at
room temperature for 0.5 to 1.0 h. The solution was neutralized by
the addition of 1M NaOH, yielding the deprotected HA-aldehyde
(formula XXII). HA--CO--R--CH(OCH.sub.3).sub.2
(XXI).fwdarw.HA--CO--R--CHO (XXII)
Example 4
[0072] Preparation of Diaminoethane-HA (formula XXIII)--Sodium
hyaluronate (100 mg, 0.25 mmol) and 1,2-diaminoethane HCl (0.998 g,
7.5 mmol) was dissolved in H.sub.2O (40 ml, 2.5 mg/ml HA). The pH
was adjusted to 6.5 and HOBT (169 mg, 1.25 mmol) predissolved in a
1:1 mixture of water and DMSO (1 ml) and EDC (240 mg, 1.25 mmol)
was added and the reaction mixture was stirred overnight. The pH
was subsequently adjusted to 7.0 with 1M NaOH and NaCl added to
produce a 5% w/v solution. HA was precipitated by addition of three
volume equivalents of ethanol. The precipitate was redissolved in
H.sub.2O at a concentration of approximately 5 mg/ml and the
precipitation repeated twice. The purified product was freeze dried
and kept at 4.degree. C. under N.sub.2. ##STR18##
Example 5
[0073] Preparation of L-Lysine methyl ester-HA (formula
XXIV)--Sodium hyaluronate (100 mg, 0.25 mmol) and L-lysine methyl
ester dihydrochloride (1.748 g, 7.5 mmol) was dissolved in H.sub.2O
(40 ml, 2.5 mg/ml HA). The pH was adjusted to 6.5 and HOBT (169 mg,
1.25 mmol) predissolved in a 1:1 mixture of water and DMSO (1 ml)
and EDC (240 mg, 1.25 mmol) was added and the reaction mixture was
stirred overnight. The pH was subsequently adjusted to 7.0 with 1M
NaOH and NaCl added to produce a 5% w/v solution. HA was
precipitated by addition of three volume equivalents of ethanol.
The precipitate was redissolved in H.sub.2O at a concentration of
approximately 5 mg/ml and the precipitation repeated twice. The
purified product was freeze dried and kept at 4.degree. C. under
N.sub.2. See FIG. 3C for NMR data of the product. ##STR19##
[0074] Example 6
[0075] Preparation of L-Histidine methyl ester HA (formula
XXV)--Sodium hyaluronate (100 mg, 0.25 mmol) and L-histidine methyl
ester dihydrochloride (1.815 g, 7.5 mmol) was dissolved in H.sub.2O
(40 ml, 2.5 mg/ml HA). The pH was adjusted to 6.5 and HOBT(169 mg,
1.25 mmol) predissolved in a 1:1 mixture of H.sub.2O and DMSO (1
ml) and EDC (240 mg, 1.25 mmol) was added and the reaction mixture
was stirred overnight. The pH was subsequently adjusted to 7.0 with
1M NaOH and NaCl added to produce a 5% w/v solution. HA was
precipitated by addition of three volume equivalents of ethanol.
The precipitate was redissolved in H.sub.2O at a concentration of
approximately 5 mg/ml and the precipitation repeated twice. The
purified product was freeze dried and kept at 4.degree. C. under
N.sub.2. See FIG. 3B for NMR data of the product. ##STR20##
Example 7
[0076] Preparation of Hydrazido-HA (formula XXVI)--Sodium
hyaluronate (100 mg, 0.25 mmol) and dihydrazide i.e. adipic
dihydrazide (1.31 g, 7.5 mmol) was dissolved in H.sub.2O (40 ml,
2.5 mg/ml HA). The pH was adjusted to 6.5 and HOBT (169 mg, 1.25
mmol) predissolved in a 1:1 mixture of water and DMSO (1 ml) and
EDC (240 mg, 1.25 mmol) was added and the reaction mixture was
stirred overnight. The pH was subsequently adjusted to 7.0 with 1 M
NaOH and NaCl added to produce a 5% w/v solution. HA was
precipitated by addition of three volume equivalents of ethanol.
The precipitate was redissolved in H.sub.2O at a concentration of
approximately 5 mg/ml and the precipitation repeated twice. The
purified product was freeze dried and kept at 4.degree. C. under
N.sub.2. See FIG. 3D for NMR data of the product. ##STR21##
Example 8
[0077] Preparation of Diaminoalkyl-HA (formula XXVII)--Sodium
hyaluronate (100 mg, 0.25 mmol) and a diaminoalkane, i.e.
1,2-diaminobutane HCl (1.208 g, 7.5 mmol) was dissolved in H.sub.2O
(40 ml, 2.5 mg/ml HA). The pH was adjusted to 7.5 and
NHS.S0.sub.3Na (268 mg, 1.25 mmol) and EDC (240 mg, 1.25 mmol) was
added and the reaction mixture was stirred overnight. The pH was
subsequently adjusted to 7.0 with 1M NaOH and NaCG added to produce
a 5% w/v solution. HA was precipitated by addition of three volume
equivalents of ethanol. The precipitate was redissolved in H.sub.2O
at a concentration of approximately 5 mg/ml and the precipitation
repeated twice. The purified product was freeze dried and kept at
4.degree. C. under N.sub.2. See FIG. 3A for NMR data of the
product. ##STR22##
Example 9
[0078] Formation of crosslinked HA hydrogels--The general procedure
for forming crosslinked HA hydrogels is as follows: Modified HA is
dissolved by agitation in H.sub.2O or phosphate buffered saline (pH
7.4-8.5) at a concentration of 5-25 mg/ml. The degree of
modification of the HA derivative is derived from the integration
of the .sup.1H NMR peaks. After complete dissolution, the HA
derivative solution is transferred to a 1 ml syringe. When reacting
the HA derivatives with low molecular weight crosslinkers, a slight
excess of the compound (about 1.1 molar equivalent of functional
groups) is dissolved in a second 1 ml syringe in 1/10 of the HA
derivative volume immediately prior to use. The syringes are
connected while paying special attention to excluded air, the
contents are rapidly mixed, typically with 20 passages, and then
extruded. When reacting HA derivative molecules with different
functionalities, 0.5-1.0 equivalent of HA-aldehyde is mixed with 1
equivalent of HA-hydrazine, depending on the degree of modification
of the HA derivatives. At room temperature, gelation occurs within
about 30 seconds to several minutes, depending on the formulation,
and the gel properties do not significantly change after
approximately 5 minutes.
Example 10
[0079] Digestion of crosslinked HA hydrogels with
hyaluronidase--The general procedure for digestion of crosslinked
HA hydrogels is as follows: HA hydrogels are formed in 1 ml
syringes by crosslinking 12 mg/ml HA-amnine in phosphate buffered
saline with various crosslinkers as indicated in FIG. 4. Gelling is
allowed to occur for 1 hour at 37.degree. C. for the reaction to be
complete, after which identical .about.100 .mu.l cylindrical gels
are formed by cutting the syringes with a razor blade. The gels are
incubated with different concentrations of bovine testicular
hyaluronidase (Sigma) 50-5000 U/mL in 400 .mu.l of 30 mM citric
acid, 150 mM Na.sub.2HPO.sub.4, pH 6.3, 150 mM NaCl for the
indicated time 0-48 hours. Degradation of the gels is determined
from the release of glucuronic acid into the supernatant as
measured using the carbazole method (Bitter and Muir, supra). See
FIG. 4.
Example 11
[0080] Crosslinked HA hydrogels as a matrix for cell
culture--Chondrocytes were isolated from bovine nose cartilage
according to established procedures (Hauselmann et al., Matrix 12,
116-129 (1992; Kuttner et al., J. Cell Biol. 93, 743-750 (1982)),
cultured in Ham's F12 medium containing 5% fetal bovine serum and
antibiotics, and dedifferentiated by monolayer culture on plastic.
For cytotoxicity studies, cells (2.5.times.10.sup.5) were embedded
into the HA hydrogels by gently mixing the trypsinized cells (
about 50 to 100 .mu.l) with the polymerizing HA and crosslinker
mixture (approximately 400 .mu.l gel volume) prior to complete
setting. Agarose embedded cells served as a control. After
adaptation to the culture conditions (24 h), cell proliferation and
metabolic activity was assessed by pulse labeling with
[.sup.3H]thynfidine and [.sup.35S]methionine. For cell infiltration
studies, HA hydrogels were polymerized in 24-well plates
(.about.15mm diameter and 3mm height) for 1 h at room temperature,
and extensively rinsed with phosphate buffered saline. Cell
adhesion molecules or chemotactic factors, e.g. IGF-1, were added
to the HA solution prior to crosslinking when desired. After 24 h,
cells (2.5.times.10.sup.5) were seeded on top of the HA-hydrogels
and cultured as above. At different time points post seeding, gels
were fixed in phosphate buffered 4% paraformaldehyde and processed
for paraffin embedding. Cell infiltration was assessed by staining
sections with Haematoxylin/Eosin. See FIG. 5.
Example 12
[0081] Subcutaneous implantation of HA hydrogels in rats--Rats (2-3
per test material) were anaesthetized with ketamine/xylazine, the
ventral thorax and abdomen shaved, and prepared aseptically. A
small vertical incision was made on either side of the xiphoid
cartilage of the sternum and the skin undermined with a blunt
instrument to separate the skin from the underlying tissue. HA
hydrogels were polymerized in 3 ml syringes as described. For
induction of chondro-osseous differentiation, 1 mg/ml
prefibrillized intact collagen type I (Organogenesis, Canton,
Mass.), 200 .mu.g/ml recombinant BMP-2 (Genetics Institute,
Cambridge, Mass.), and 500 ng/ml IGF-1 (Celtrix Pharmaceuticals,
Santa Clara, Calif.) or 50 ng/ml TGF-.beta.2 (Celtrix
Pharmaceuticals, Santa Clara, Calif.) were mixed with the HA
solution prior to crosslinking. Collagen fibrils were prepared by
slow polymerization (from dilute solutions of 2-3 mg/ml) of
acid-solubilized couagen in phosphate buffered saline and harvested
by centrifugation following standard protocols (McPherson et al.,
Collagen Rel. Res. 5, 119-135 (1985)). Gelling of the HA hydrogels
was allowed to occur for 24 h at room temperature for the reaction
to be complete, after which identical .about.3mm thick cylindrical
gels were prepared by cutting the syringes with a razor blade. HA
hydrogel discs were then placed in each pocket and the skin
incisions closed with sutures. Ten days post operatively, the rats
were euthanized and the appearance of the implant sites, i.e.
degree of inflammation, grossly examined and tissue specimens
harvested and processed for histology by fixation in phosphate
buffered formalin and paraffin embedding. Sections were stained
with Haematoxylin/Eosin and with Safranin-O/fast green, and cell
infiltration and transformation (cartilage and bone formation)
induced by the biomaterial as well as signs of fibrosis and
inflammation in the surrounding tissue evaluated. See FIGS.
6-8.
* * * * *