U.S. patent application number 15/621627 was filed with the patent office on 2017-12-14 for glycosaminoglycan-based materials as an engineered biocompatible cellular matrix.
This patent application is currently assigned to Purdue Research Foundation. The applicant listed for this patent is Purdue Research Foundation. Invention is credited to Gert J. Breur, Meng Deng, Liangju Kuang.
Application Number | 20170354758 15/621627 |
Document ID | / |
Family ID | 60573558 |
Filed Date | 2017-12-14 |
United States Patent
Application |
20170354758 |
Kind Code |
A1 |
Deng; Meng ; et al. |
December 14, 2017 |
Glycosaminoglycan-Based Materials as an Engineered Biocompatible
Cellular Matrix
Abstract
Disclosed herein is a cross-linked polymeric system comprising
thiolated hyaluronic acid (HA), thiolated chondroitin sulfate (CS),
and functionalized polyethylene glycol (PEG), wherein said
functionalized PEG cross-links thiolated HA and thiolated CS.
Methods of fabrication and utilization of the same are also
claimed. This polymeric system may be used as an engineered
biocompatible cellular matrix for 3D cell culture, tissue
engineering and regenerative medicine applications.
Inventors: |
Deng; Meng; (West Lafayette,
IN) ; Kuang; Liangju; (West Lafayette, IN) ;
Breur; Gert J.; (West Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation |
West Lafayette |
IN |
US |
|
|
Assignee: |
Purdue Research Foundation
West Lafayette
IN
|
Family ID: |
60573558 |
Appl. No.: |
15/621627 |
Filed: |
June 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62349475 |
Jun 13, 2016 |
|
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|
62366160 |
Jul 25, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/485 20130101;
A61L 2300/64 20130101; C08F 251/00 20130101; A61L 2300/414
20130101; A61L 27/3834 20130101; C08L 67/04 20130101; C08L 5/08
20130101; A61L 27/26 20130101; A61L 27/20 20130101; A61L 27/18
20130101; A61L 27/52 20130101; A61L 2430/02 20130101; A61L 2430/06
20130101; A61K 47/61 20170801; A61K 9/0019 20130101; A61L 27/18
20130101; A61L 27/3817 20130101; A61K 47/36 20130101; A61L 27/26
20130101; A61L 27/54 20130101; A61K 9/06 20130101 |
International
Class: |
A61L 27/20 20060101
A61L027/20; A61L 27/54 20060101 A61L027/54; A61L 27/18 20060101
A61L027/18; A61L 27/38 20060101 A61L027/38; A61L 27/52 20060101
A61L027/52 |
Claims
1. A composition comprising thiolated hyaluronic acid (HA),
thiolated chondroitin sulfate (CS) and a functionalized
polyethylene glycol (PEG) derivative, wherein said PEG derivative
crosslinks thiolated HA and thiolated CS.
2. The composition of claim 1 wherein said functionalized PEG
derivative contains a plurality of activated vinyl groups.
3. The composition of claim 1 wherein the molecular ratio of the
total thiol group of HA and CS vs. said activated vinyl group is
maintained constantly as about 1.07.
4. The composition of claim 1 wherein said activated vinyl groups
are selected from the group consisting of poly (ethylene glycol)
diacrylate (PEGDA), poly (ethylene glycol) Divinyl Sulfone (PEGVS),
and 4-arm poly (ethylene glycol) vinyl Sulfone (4PEGVS).
5. The composition of claim 1 wherein thiolated HA further
comprising a plurality of amino groups (--NH.sub.2) groups selected
from the following formula I-IV: ##STR00002## wherein the
functional groups R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5
comprise any one of or a combination of haloacetates, dihydrazides,
amines, thiols, carboxylic acids, aldehydes, ketones, active
hydrogen sites on aromatic ring, dienes, azide isothiocyanates,
isocyanates, acyl azides, NHS esters, sulfo-NHS, sulfonyl chloride,
epoxides, carbonates, aryl halides, imidoesters, carbodiimides
(e.g. DCC and EDC), alkylphosphate compounds, anhydrides,
fluorophenyl esters, hydroxymethyl phosphines, guanidino groups,
iodoacetyl derivatives, maleimides, aziridines, acryloyl
derivatives, arylating agents, disulfide derivatives, vinylsulfone,
phenylthioester, cisplatins, diazoacetates, carbonyl diimidazoles,
oxiranes, N, N'-disuccinimidyl carbonates, N-hydroxylsuccinimidyl
chloroformates, alkyl halogens, hydrazines, alkynes, and
phosphorus-bound chlorine.
6. An osteochondral regenerative engineering composite comprising
poly (lactide-co-glycolide) (PLGA) grafted to a composition via
--NH.sub.2 group to form a bone mimetic, wherein said composition
comprising thiolated hyaluronic acid (HA), thiolated chondroitin
sulfate (CS) and a functionalized polyethylene glycol (PEG)
derivative, wherein said PEG derivative crosslinks thiolated HA and
thiolated CS.
7. The osteochondral regenerative engineering composite of claim 6
wherein said PLGA is made of lactic acid/glycolic acid at a ratio
of about 85:15.
8. The composition of claim 1 wherein the molecular weight and
length of said PEG derivatives are adjustable to modify the
composite modular storage and loss value.
9. The composition of claim 8, wherein the molecular weight of said
PEG derivatives ranges from about 700 Da to about 8000 Da.
10. A method of making a composition of cross-linked HA, CS and PEG
comprising the steps of: preparing thiolated HA; a. preparing
thiolated CS; b. preparing PEG derivative containing a plurality of
activated functionalities; c. mixing said HA, CS, and PEG
derivative in an aqueous medium; and d. initiating cross-linking;
wherein said activated functionality is elected from the group
consisting of alkoxysulfonate, arylsulfonate, heteroarylsulfonate,
maleimido, ether NHS esters, sulfo-NHS, wherein the structures of
said PEG derivative is any one of or a combination of linear,
dendrimers-like, star-shaped, hyper-branched, combed, brushed,
cross-linked architectures, fibers, microspheres, and
nanoparticles.
11. The method of claim 10, wherein the activated functionality of
PEG comprises any one of or a combination of isothiourea, isourea,
amide, sulfonamide, secondary amine, sulfonamide, shift-base,
secondary amino-methyl, carbamate, aryl amine, amidine, amide,
phosphoramidate, guanidine, substituted imidocarbonate, thioether,
4-amino derivative of cytosine, aryl thioether, disulfide,
sulfonate, .beta.-thiosulfonyl, ester, carbamate, hydrazone, diazo,
triazoles, iodinated compound, carbohydrates, amino acid esters
bond, cycloalkene, oxime triazole, and triazoline.
12. The composition of claim 1 further comprising functionalized
peptides selected from the group consisting of
arginine-glycine-aspartate (RGD), fibronectin, laminin, and
fibrinogen, wherein said peptides are functionalized by carboxyl,
amine or thiol group, and conjugated with HA, CS, or functionalized
PEG derivatives through thiol-ene click reactions and
esterification.
13. The composition of claim 1 further comprising tissue
engineering cells, wherein the cells are selected from the group
consisting of mesenchymal stem cells, osteoblast, chondrocytes,
adipocyte, fibroblast, hepatocytes, enterocytes, urothelial cells,
blood cells, skin cells, endothelial cells, nerve cells, sex cells,
cancer cells and combination thereof.
14. The composition of claim 1 further comprising small molecules
as therapeutic agents,
15. The composition of claim 1 further comprising at least one
growth factor.
Description
CROSS REFERENCE
[0001] This application claims the benefits of U.S. provisional
application 62/349,475, filed on Jun. 13, 2016 and U.S. provisional
application 62/366,160, filed on Jul. 25, 2016. The content of
which are expressly incorporated herein entirely.
TECHNICAL FIELD
[0002] The present disclosure generally relates to engineered
biocompatible cellular matrices, and in particular to polymeric
systems that may be used as engineered biocompatible cellular
matrices for 3D cell culture, tissue engineering and regenerative
medicine applications.
BACKGROUND
[0003] This section introduces aspects that may help facilitate a
better understanding of the disclosure. Accordingly, these
statements are to be read in this light and are not to be
understood as admissions about what is or is not prior art.
[0004] A biomaterial-based three-dimensional (3D) cell
microenvironment (niche) is often designed to mimic a natural
extracellular matrix (ECM) and support cell growth,
differentiation, and organization to form regenerated tissue[1].
Hydrogels composed of polymer networks swollen in water provide an
attractive biomaterial platform to provide ECM-mimicking and
cell-interactive properties[2]. The regenerative efficacy is
greatly dependent on precise control of material properties and
optimization of cell-material interactions. For example,
mesenchymal stem cells (MSCs) can sense the differences in
mechanical environments such as stiffness and initiate the
mechanotransduction pathways, which ultimately affect cell fate and
distinct lineage differentiation[3, 4].
[0005] Glycosaminoglycans (GAGs) constitute an important part of
the ECM. GAGs may be divided into hyaluronic acid (HA) and sulfated
GAGs including chondroitin sulfate (CS), dermatan sulfate (DS),
keratan sulfate (KS), heparin sulfate, and heparan sulfate (HS). HA
is not linked to proteins, while sulfated GAGs in nature are
covalently linked to proteins. HA is an immunoneutral
polysaccharide that includes alternating disaccharide units of
[.beta.(1,4)-D-glucuronic acid-.beta.(1,3)-N-acetyl-D-glucosamine)
linkages[5] and is widely distributed throughout the body,
especially in the synovia of joints, the corpus vitreum of the
eyes, and the dermis of the skin. HA is predominantly localized to
the extracellular and pericellular matrix. Functionally, HA
contributes to the elastoviscosity of fluid connective tissues
including synovial fluid and vitreous humor, and it modulates
hydration and transport of water through tissues[6]. HA also plays
an important role in many biological processes including
facilitating wound healing[7], regulating cell adhesion and
proliferation[8], cell motility, angiogenesis, cellular signaling,
and matrix organization[9]. The enzymatic degradation of HA results
from the action of three types of enzymes: hyaluronidase,
.beta.-D-glucuronidase, and .beta.-N-acetyl-hexosaminidase.[10].
Inherently biocompatible and biodegradable[5], HA has been used
clinically for more than thirty years[5, 11, 12]. For example, it
has been approved by the US Food and Drug Administration for the
treatment of osteoarthritis in humans since 1997[13]. Therefore,
hyaluronic acid (HA) has attracted significant research interests
to develop hydrogels because it offers both a biomimetic
microenvironment found in natural ECM and the availability of
carboxylic acids and hydroxyl groups necessary for
modification[5].
[0006] Chondroitin sulfate (CS), a polysaccharide composing of
alternating units of .beta.-1, 4-linked glucuronic acid and
.beta.-1, 3-Nacetylgalactosamine, is sulfated on either the 4 or 6
position of the galactosamine residues. CS is the major
glycosaminoglycan (GAG) in the ECM of all vertebrates and is found
in bone, cartilage, and connective tissues[14]. GAGs in normal
human meniscal tissues include 60% chondroitin-6-sulfate and 10-20%
chondroitin-4-sulfate[15]. CS is an appealing material in tissue
regeneration due to its biological implications in supporting
cellular activities in the ECM[16, 17], as well as
anti-inflammatory and wound healing properties[4,18, 19].
[0007] However, hydrogels derived from either HA or CS suffer from
limited mechanical properties and uncontrollable degradation rates,
which necessitates the need to combine GAGs with other polymers
[20, 21]. Here, we developed a novel GAG-based hydrogel system as a
tunable cell niche through combination of functional
polysaccharides and a cross-linker. GAG hydrogels may be used for
tissue engineering due in part to their ability to efficiently
encapsulate cells. Mechanical and structural properties, and
cell-material interactions may be manipulated by modification of
the crosslinking density and structures of polysaccharides and a
cross-linker. In some cases, cross-linked polymers or gels may have
a high, tissue-like water content, which may allow nutrient and
waste transport.
SUMMARY
[0008] This disclosure provides a composition comprising thiolated
hyaluronic acid (HA), thiolated chondroitin sulfate (CS) and
functionalized polyethylene glycol (PEG) derivative, wherein said
PEG derivative cross-links thiolated HA and thiolated CS.
[0009] In some preferred embodiment, the aforementioned
functionalized PEG derivative contains a plurality of activated
vinyl groups.
[0010] In some embodiment the aforementioned composition has a
constant molecular ratio of the total thiol group of HA and CS vs.
the activated vinyl group. In some preferred embodiment, the
molecular ratio of the total thiol groups vs. the activated vinyl
group is about 1.07.
[0011] In some preferred embodiment, the aforementioned activated
vinyl group is selected from the group consisting of poly (ethylene
glycol) diacrylate (PEGDA), poly (ethylene glycol) Divinyl Sulfone
(PEGVS), and 4-arm poly (ethylene glycol) vinyl Sulfone
(4PEGVS).
[0012] In some preferred embodiment the aforementioned thiolated HA
further comprising a plurality of amino groups (--NH.sub.2)
selected from the following formula I-IV:
##STR00001##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 may include
any one of or a combination of haloacetates, dihydrazides, amines,
thiols, carboxylic acids, aldehydes, ketones, active hydrogen sites
on aromatic ring, dienes, azide isothiocyanates, isocyanates, acyl
azides, N-hydroxysuccinimide (NHS) esters, sulfo-NHS, sulfonyl
chloride, epoxides, carbonates, aryl halides, imidoesters,
carbodiimides (e.g. N, N'-dicyclohexylcarbodiimide (DCC) and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)),
alkylphosphate compounds, anhydrides, fluorophenyl esters,
hydroxymethyl phosphines, guanidino groups, iodoacetyl derivatives,
maleimides, aziridines, acryloyl derivatives, arylating agents,
disulfide derivatives, vinylsulfone, phenylthioester, cisplatins,
diazoacetates, carbonyl diimidazoles, oxiranes, N,
N'-disuccinimidyl carbonates, N-hydroxylsuccinimidyl
chloroformates, alkyl halogens, hydrazines, alkynes, and
phosphorus-bound chlorine.
[0013] This disclosure further provides an osteochondral
regenerative engineering composite comprising poly
(lactide-co-glycolide) (PLGA) grafted to aforementioned composition
via --NH.sub.2 group to form a bone mimetic.
[0014] In some preferred embodiment, the aforementioned PLGA is
made of lactic acid/glycolic acid at a ratio of about 85:15.
[0015] In some preferred embodiment, the molecular weight and
length of aforementioned PEG derivatives are adjustable to modify
the composite modular storage and loss value.
[0016] In some preferred embodiment, the aforementioned PEG
derivatives have molecular weight ranging from about 700 Da to
about 8000 Da.
[0017] This disclosure further discloses a method of making a
composition of cross-linked HA, CS and PEG, the method comprising
the steps of: [0018] a. preparing thiolated HA; [0019] b. preparing
thiolated CS; [0020] c. preparing PEG derivative containing a
plurality of activated functionalities; [0021] d. mixing said HA,
CS, and PEG derivative in an aqueous medium to crosslink HA, CS,
[0022] wherein the activated functionality is selected from the
group consisting of alkoxysulfonate, arylsulfonate,
heteroarylsulfonate, maleimido, ether NHS esters, and sulfo-NHS,
[0023] wherein the structures of the cross-linker comprise any one
of or a combination of linear, dendrimers-like, star-shaped,
hyper-branched, combed, brushed, cross-linked architectures,
fibers, microspheres, and nanoparticles.
[0024] In some preferred embodiment, the aforementioned activated
functionalities of PEG comprises any one of or a combination of
isothiourea, isourea, amide, sulfonamide, secondary amine,
sulfonamide, shift-base, secondary amino-methyl, carbamate, aryl
amine, amidine, amide, phosphoramidate, guanidine, substituted
imidocarbonate, thioether, 4-amino derivative of cytosine, aryl
thioether, disulfide, sulfonate, .beta.-thiosulfonyl, ester,
carbamate, hydrazone, diazo, triazoles, iodinated compound,
carbohydrates, amino acid esters bond, cycloalkene, oxime triazole,
and triazoline.
[0025] In some of the preferred embodiment, the aforementioned
engineered composite further comprising peptides selected from the
group consisting of arginine-glycine-aspartate (RGD), fibronectin,
laminin, and fibrinogen, wherein said peptides comprising
amine-functionalized laminin, carboxyl functionalized laminin,
amine functionalized RGD, and thiolated RGD. These peptides can be
conjugated with HA, CS, or functionalized PEG (e.g., PEGDA, PEGDVS,
4PEGVS) through thiol-ene click reactions and esterification.
[0026] In some of preferred embodiment, the aforementioned
composition further comprising cells selected from the group
consisting of mesenchymal stem cells, osteoblasts, chondrocytes,
adipocytes, fibroblasts, hepatocytes, enterocytes, urothelial
cells, blood cells, skin cells, endothelial cells, nerve cells, sex
cells, cancer cells and combination thereof.
[0027] In some preferred embodiment, the aforementioned composition
further comprising small molecules as therapeutic agents.
[0028] In some preferred embodiment, the aforementioned composition
further comprising at least one growth factor.
[0029] These and other features, aspects and advantages of the
present invention will become better understood with reference to
the following figures, associated descriptions and claims.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1 shows the formation of GAG-based polymeric systems
via covalent or non-covalent interactions.
[0031] FIG. 2 shows functionalized monomeric hyaluronic acid
represented by formula I, II, III, IV.
[0032] FIG. 3 shows examples of covalent chemistries for
crosslinking functionalized HA with a cross-linker.
[0033] FIG. 4 shows schematic and structural representations
showing cross-linker structures.
[0034] FIG. 5 shows an example of non-covalent chemistries for
crosslinking functionalized HA with a cross-linker.
[0035] FIG. 6 shows a schematic illustration of design of
bio-inspired composite hydrogels as an engineered cell niche via
thiol-ene click chemistry. Modulation of hydrogel properties
enables regulation of cellular behavior.
[0036] FIG. 7 shows a biomimetic Hydrogel Formation. Carboxyl
groups in HA or CS were reacted with cystamine dihydrochloride by
activation with EDC and NHS. Reduction of cystamine by DTT resulted
in free thiol groups in the HA or CS backbone. Crosslinking of
HA-SH and CS--SH mixtures by conjugate addition using PEGDA.
[0037] FIG. 8. (FIG. 8A).sup.1H NMR spectrum confirms successful
synthesis of HA-conjugated cystamines. Compared to the spectrum of
HA (the N-acetyl methyl proton of HA were at .delta. 1.95), new
resonances appeared at .delta. 2.33, 2.54, 2.77 and 2.90
corresponding to methylenes (--CH.sub.2CH.sub.2--). (FIG.
8B).sup.1H NMR spectrum confirms successful synthesis of
CS-conjugated cystamines. Compared to the spectrum of CS (the
N-acetyl methyl proton of CS were at .delta. 1.95), new resonances
appeared at .delta. 2.25, 2.55, 2.77 and 2.90 corresponding to
methylenes (--CH.sub.2CH.sub.2--).
[0038] FIG. 9. (FIG. 9A).sup.1H NMR spectrum confirms successful
synthesis of HA-SH. (FIG. 9B).sup.1H NMR spectrum confirms
successful synthesis of CS--SH.
[0039] FIG. 10. (FIG. 10A) The inversion of a hydrogel. (FIG. 10B)
Hydrogels in cell culture.
[0040] FIG. 11 shows Cryo SEM images of (FIG. 11A, FIG. 11D) HCP
700, (FIG. 11B, FIG. 11E) HCP 3400, and (FIG. 11C, FIG. 11F)
HCP8000 hydrogels.
[0041] FIG. 12 shows tunable HCP hydrogel properties. (FIG. 12A)
Gelation time. (FIG. 12B) Swelling ratio. (FIG. 12C) FITC-dextran
(70k) release from hydrogels as a function of time at pH 7.4,
37.degree. C. demonstrating the potential to achieve tunable
release profiles of macromolecules.
[0042] FIG. 13 shows tunable mechanical properties. (FIG. 13A)
Strain sweeps of hydrogel HCP700L. The linear-viscoelastic limit
was determined with respect to strain. Storage modulus (G') was
determined from 0.01 to 10% strain. (FIG. 13B) Frequency sweeps of
hydrogel HCP700L. The linear modulus plateau (G') with respect to
frequency was determined. (FIG. 13C) Time evolution of hydrogel
HCP700L, HCP3400L, HCP8000L with time at 37.degree. C. (FIG. 13D)
The rheological properties for hydrogels. Increasing the chain
length of cross-linker (PEGDA) results in a stiffer gel.
[0043] FIG. 14 shows 3D encapsulation procedures.
[0044] FIG. 15. Live/Dead assay of hMSCs encapsulated in hydrogels
after 1 day of culture (FIG. 15A, FIG. 15D) HCP700 (FIG. 15B, FIG.
15E) HCP3400 (FIG. 15C, FIG. 15F) HCP8000 hydrogels.
[0045] FIG. 16. Live/Dead assay of hMSCs encapsulated in HCP3400
hydrogels after (FIG. 16A, FIG. 16C) 1 day of culture (FIG. 16B,
FIG. 16D) 5 days of culture.
[0046] FIG. 17 shows actin staining of hMSCs encapsulated in
hydrogels after 1 day of culture; Actin, red; nucleus, blue. (FIG.
17A) HCP700 (FIG. 17B) HCP3400 (FIG. 17C) HCP8000 hydrogels.
[0047] FIG. 18 shows stiffened hydrogels exhibiting increased focal
adhesion kinase phosphorylation of hMSCs as evidenced from FLIM.
FAK is an early mechanosensor that responds to changes in substrate
stiffness.
[0048] FIG. 19. (FIG. 19A) SEM image of dehydrated hydrogel
HCP8000, 200 .mu.m scale. Cell deposited matrix visible on surface.
(FIG. 19B) SEM image of dehydrated hydrogel HCP8000 showing cell
deposited matrix on surface, 50 .mu.m scale. (FIG. 19C) SEM image
of dehydrated hydrogel HCP8000 showing a porous structure
appropriate for cell migration, 50 .mu.m scale.
[0049] FIG. 20. Comparison of PEGVS and PEGDA. (FIG. 20A) Time
evolution of hydrogel HCPVS3400 with time at 37.degree. C. The
gelation time was 6.2 min. (FIG. 20B) Storage modulus at the
equilibrium stage. Due to more reactive vinyl sulfone moiety, PEGVS
demonstrates significantly higher storage modulus compared to
PEGDA.
[0050] FIG. 21. In vitro study. (FIG. 21A) Live/Dead assay of
primary chondrocytes encapsulated in HCPV3400 hydrogels after 21
day of culture. (FIG. 21B) Primary chondrocytes were isolated
freshly from pig. At P2 stage, cells were encapsulated in the
hydrogel and cultured for 21 days with DMEM supplemented with 20%
FBS. Cells were labeled with actin (red) and aggrecan (green).
Nuclei were counterstained with DAPI (blue).
[0051] FIG. 22. Mechanical properties of HA/CS/4 arm PEG (HC4PVS).
(FIG. 22A) Time evolution of hydrogel HC4PVS20000 with time at
37.degree. C. The gelation time was 12 seconds. (FIG. 22B) Storage
modulus at the equilibrium stage of hydrogel HC4PVS20000 at day 1
and day 40.
[0052] FIG. 23. (FIG. 23A) Live/Dead assay (FIG. 23B) Actin
staining of hMSCs encapsulated in HC4PVS20000 hydrogels after 21
days of culture. Actin, red; nucleus, blue.
[0053] FIG. 24. Design of an integrated scaffold system for
treatment of osteochondral defects via in situ regeneration of
bone, cartilage and the bone-cartilage interface.
[0054] FIG. 25. SEM images of (FIG. 25A, FIG. 25C) PLGA 3D
scaffolds. (FIG. 25B, FIG. 25D) PLGA 3D scaffold-hydrogel
integration.
[0055] FIG. 26. Raman spectra of the HA-SH, CS--SH, PEGDA700, and
lyophilized HCP700 hydrogel at 0 min and 60 min.
[0056] FIG. 27. Physical characterization of hydrogels: (FIG. 27A)
Plateau of G'. (FIG. 27B) Gelation times determined by the vial
tilting method in PBS at 37.degree. C. (FIG. 27C) Time sweep
showing storage (G') and loss (G'') with increasing MW of
crosslinker and high thiol degrees of substation (i.e., HCP700,
HCP3400 and HCP8000 hydrogels).
[0057] FIG. 28. Dynamic FAK phosphorylation dependents on hydrogel
stiffness. (FIG. 28A) Schematic illustration of real time
measurement of FAK phosphorylation in hMSCs laden hydrogels. (FIG.
28B) Quantitative analysis showed average fluorescence lifetime for
hydrogels (n=26-50) with different pleatu G'. (FIG. 28C)
Quantitative analysis shows FAKSOR average lifetime in hMSC-laden
HCP700L (n=20) and HCP700 hydrogels (n=50). At the same as total
polymer percentage and MW of PEGDA, HCP 700L use low thiol
substitution degree of HA-SH and CS--SH and HCP700 utilized high
thiol substitution degree of HA-SH and CS--SH.
[0058] FIG. 29. (FIG. 29A) Stiffened hydrogels exhibiting increased
FAK phosphorylation of hMSCs encapsulated in 3400 hydrogels (n=3).
(FIG. 29B) FAKSOR average lifetime in hMSC-laden HCP3400 hydrogels
remain constant over 30 minutes right after G' reached plateau
(n=5).
[0059] FIG. 30. Quantitative Real Time PCR for (Bone morphogenetic
protein 2) (BMP2), alkaline phosphatase (ALP), collagen (CoII), and
osteonectin (ON) expression in the hydrogels with PEG700DA or
PEGDA34000 as a crosslinker after 5 days in growth medium.
[0060] FIG. 31. hMSCs encapsulated in hydrogels after 3 day of
culture with RGD concentration of (FIG. 31A) 0.25 mM, (FIG. 31B)0.5
mM and (FIG. 31C) 1 mM. Scale bar: 10 .mu.m.
DETAILED DESCRIPTION
[0061] For the purposes of promoting an understanding of the
principles of the present disclosure, reference will now be made to
the embodiments illustrated in the drawings, and specific language
will be used to describe the same. It will nevertheless be
understood that no limitation of the scope of this disclosure is
thereby intended.
[0062] While the concepts of the present disclosure are illustrated
and described in detail in the figures and the description herein,
results in the figures and their description are to be considered
as exemplary and not restrictive in character. It is understood
that only the illustrative embodiments are shown and described and
that all changes and modifications that come within the spirit of
the disclosure are desired to be protected.
[0063] Unless defined otherwise, the scientific and technology
nomenclatures have the same meaning as commonly understood by a
person in the ordinary skill in the art pertaining to this
disclosure.
[0064] As disclosed herein, in at least one embodiment, a GAG-based
polymeric system can be prepared by following two strategies: (i)
chemical covalent bonding and (ii) physically entrapped and/or
entangled (non-covalent) strategies as shown in FIG. 1.
[0065] In at least one embodiment, GAG polysaccharides can be
modified in predictable synthetic routes to control the properties
of the resulting materials, including modifications leading to
hydrophobicity and biological activities. In part, the present
disclosure provides for a composition comprising at least one
monomeric unit of HA functionalized by at least one functional
group moiety. Chemical modifications of HA can be targeted to three
functional groups: the glucuronic acid carboxylic acid, the primary
and secondary hydroxyl groups, and the N-acetyl group (following
deamidation). In some embodiments, compositions of HA in the
present disclosure are provided that may be represent by Formula I,
II, III and IV (FIG. 2).
[0066] Carboxylates in a HA backbond can be modified by
carbodiimide-mediated reactions, esterification, and amidation.
Hydroxyls in a HA backbond can be modified by etherification,
divinylsulfone crosslinking, esterification, and bisepoxide
crosslinking. Additionally, converting diols to aldehydes can be
achieved through periodate oxidation of HA. Finally, deacetylation
of the N-acetyl group of HA recovers an amino group which can then
react with an acid using the same amidation.
[0067] The functional groups R.sub.1, R.sub.2, R.sub.3, R.sub.4,
and R.sub.5 may include any one of or a combination of
haloacetates, dihydrazides, amines, thiols, carboxylic acids,
aldehydes, ketones, active hydrogen sites on aromatic ring, dienes,
azide isothiocyanates, isocyanates, acyl azides,
N-hydroxysuccinimide (NHS) esters, sulfo-NHS, sulfonyl chloride,
epoxides, carbonates, aryl halides, imidoesters, carbodiimides
(e.g. N, N'-dicyclohexylcarbodiimide (DCC) and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)),
alkylphosphate compounds, anhydrides, fluorophenyl esters,
hydroxymethyl phosphines, guanidino groups, iodoacetyl derivatives,
maleimides, aziridines, acryloyl derivatives, arylating agents,
disulfide derivatives, vinylsulfone, phenylthioester, cisplatins,
diazoacetates, carbonyl diimidazoles, oxiranes, N,
N'-disuccinimidyl carbonates, N-hydroxylsuccinimidyl
chloroformates, alkyl halogens, hydrazines, alkynes, and
phosphorus-bound chlorine.
[0068] The functionalized polysaccharides may include the
combination of HA and CS. HA may be used in an amount ranging from
0.1% to 99% by weight. HA has an average molecular weight in the
range of 10-3,000 Kilo Daltons, preferably 1000-3000 Kilo Daltons.
Sulfated GAGs may be used in an amount ranging from 0.1% to 99% by
weight.
[0069] Cross-linkers can be both synthetic polymers and natural
polymers. The natural polymers may include any one of or a
combination of fibrin, collagen, matrigel, elastin, elastin-like
peptides, albumin, natural poly (amino acids) (e.g. cyanophycin,
poly (lysine), and poly (.gamma.-glutamic acid)), polysaccharides
(e.g. chitosan, dextran, chondroitin sulfate, agarose, alginate,
methylcellulose, and heparin), .alpha.-cyclodextrin (CD),
.beta.-CD, .gamma.-CD, and blends thereof.
[0070] Synthetic polymers may include any one of or a combination
of poly (aliphatic ester) (e.g. poly(lactide) (PLA)),
poly(.epsilon.-caprolactone) (PCL), poly(glycolic acid) (PGA),
poly(lactic-co-glycolic acid) (PLGA), poly(trimethylene carbonate)
(PTMC), polydioxanone (PDS), poly(ortho ester), polyanhydrides,
poly(anhydride-co-imide), poly(anhydride-esters), polyurethanes
(e.g. degrapols), poly(amides), poly(esteramides),
poly(orthoesters), poly(dioxanones), poly(acetals), poly(ketals),
poly(carbonates), poly(orthocarbonates), poly(hydroxylbutyrates),
poly(hydroxyl-valerats), poly(alkylene oxalates), poly(alkylene
succunates), poly(malic acid), poly(amino acids),
poly(vinylpyrolidone), poly(hydroxycellulose), poly(glycerol
sebacate), poly(ethylene imine), poly(acrylic acid)(PAA), poly(N,
N'-diethylaminoethyl methacrylate) (PDEAEMA), polyethylene glycol
(PEG), poly(propylene oxide) (PPO), PEO-b-PPO block copolymers
(e.g. pluronics or poloxamers, and tetronic), poly(vinyl alcohol)
(PVA), poly(N-isopropylacrylamide) (PNIPAm),
poly(N,N-diethylacrylamide) (PDEAAm), poly(oxazolines) (e.g.
poly(2-methyloxazoline and poly(2-ethyl-2-oxazoline)),
oligo(poly(ethylene glycol) fumarates), poly(propylene fumarate),
poly(alkyl cyanoacrylates), poly(acrylic amide), synthetic
poly(amino acids) (e.g. poly (L-glutamic acid) (L-PGA) and poly
(aspartic acid)), poly(phosphazenes), poly(phosphoesters), and
blends thereof.
[0071] A cross-linker may include a homo- or hetero-functional
modifier of the following formula: A-cross-linker-Z
[0072] In some embodiment, one of A and Z is a moiety selected from
the group consisting of hydroxyls, thiols, aminos, alkyls,
alkenyls, alkoxysulfonate, arylsulfonate, heteroarylsulfonate,
azides, maleimido, propargyl, haloacetate, dihydrazide, amines,
carboxylic acids, aldehydes, ketones, active hydrogen sites on
aromatic ring, dienes, azide isothiocyanates, isocyanates, acyl
azides, ether NHS esters, sulfo-NHS, pentafluorophenyl(PFP),
azlactones, sulfonyl chloride, epoxides, carbonates, aryl halides,
imidoesters, biotin, carbodiimides (e.g. DCC and EDC),
alkylphosphate compounds, anhydrides, fluorophenyl esters,
hydroxymethyl phosphine, guanidino groups, iodoacetyl derivatives,
maleimides, aziridines, acryloyl derivatives, arylating agents,
disulfide derivatives, vinylsulfone, phenylthioester, cisplatin,
diazoacetate, carbonyl diimidazole, oxiranes, N, N'-disuccinimidyl
carbonate, N-hydroxylsuccinimidyl chloroformate, alkyl halogens,
hydrazine, maleimide, alkyne, and phosphorus-bound chlorine.
[0073] Structures of a cross-linker may be any one of or a
combination of linear, dendrimers-like, star-shaped,
hyper-branched, combed, brushed, cross-linked architectures,
fibers, microspheres, and nanoparticles. Examples are shown in FIG.
4. The number of arms in star-shaped polymers may be two or more.
Fibers may comprise any one of or a combination of polymer fibers,
carbon fibers, and ceramic fibers. Microspheres and nanoparticles
may comprise any one of or a combination of polymer
micro/nanospheres, iron oxide, silica, gold, and mesoporous silica
nanoparticles.
[0074] A cross-linker with homo- or hetero-functional groups can be
prepared by addition or chain growth polymerizations, coordination
polymerizations, and condensation or step growth polymerizations.
Addition or chain growth polymerizations include free radical
polymerization, controlled-living radical polymerization (e.g. atom
transfer radical polymerization (ATRP), reversible addition
fragmentation transfer (RAFT) polymerization, and
nitroxide-mediated radical polymerization (NMP)), cationic
polymerizations, anionic polymerizations and the like.
[0075] The conjugation of functionalized HA and cross linkers can
be achieved by any one of or the combination of the following
reactions: carbodiimide-mediated reactions, esterification,
amidation, aldehyde and ketone reactions, active hydrogen
reactions, photo-chemical reaction, azide-alkyne cycloaddition
(e.g. copper-catalyzed azide-alkyne cycloaddition (CuAAC),
copper-free azide-alkyne huisgen cycloaddition or strain-promoted
azide-alkyne cycloaddition (SPACC)), thiol-based click reaction
(thiol-yne, thiol-ene, thiol-isocyanate, thiol-Michael addition),
Diels-Alder reactions, tetrazole cycloaddition, nitrile oxide
cycloaddition, oxime/hydrazone formation, enzymatic crosslinking,
and coordination chemistry, and ligand exchange reactions. Examples
are shown in FIG. 3.
[0076] The conjugated linkages may include any one of or a
combination of isothiourea, isourea, amide, sulfonamide, secondary
amine, sulfonamide, shift-base, secondary amino-methyl, carbamate,
aryl amine, amidine, amide, phosphoramidate, guanidine, substituted
imidocarbonate, thioether, aryl thioether, disulfide, sulfonate,
thiosulfonyl, ester, ether, carbamate, hydrazone, diazo, triazoles,
iodinated compound, carbohydrates, amino acid esters bond,
cycloalkene, oxime triazole, and triazoline.
[0077] Physical interactions may include any one of or a
combination of hydrophobic interactions, hydrophilic interactions,
hydrogen bonding, electrostatic interactions, and van der Waal
interactions. Examples are shown in FIG. 5.
[0078] The GAG-based system can be used as a cellular matrix for 3D
cell culture and tissue engineering as well as a delivery vehicle
for therapeutic agents including but not limited to cells, growth
factors, and small molecules.
[0079] Cells to be encapsulated within the engineered composite may
comprise any one of or the combination of, but not limited to,
mesenchymal stem cells, osteoblast, chondrocytes, adipocyte,
fibroblast, hepatocytes, enterocytes, urothelial cells, blood
cells, skin cells, endothelial cells, nerve cells, sex cells, and
cancer cells.
EXAMPLES
Example 1: HA/CS/Poly (Ethylene Glycol) Diacrylate Composite
Hydrogel as hMSCs Niches
[0080] We developed a novel biomimetic hydrogel system as a tunable
stem cell niche through the combination of thiolated HA (HA-SH) and
thiolated CS (CS--SH) cross-linked with poly (ethylene glycol)
diacrylate (PEGDA) as shown in FIG. 6. The combination of HA and CS
offers a cell-friendly microenvironment found in native tissues,
whereas the selection of PEG is based on its established
biocompatibility and chemical versatility. The efficient control of
various hydrogel properties is demonstrated by simply varying the
molecular weight of PEG. A thiol-ene click reaction was selected
due to its high efficiency in aqueous media, reliability, lack of
by-products, unsubstantial degradation of the polysaccharide
backbone, and without the use of any metal catalysts.sup.[22]. To
our knowledge, this is the first report that tricopolymers of HA,
CS, and PEG in the forms of natural/synthetic composite hydrogels
were fabricated through thiol-ene click chemistry. We showed the
efficient control of various hydrogel properties (e.g., swelling,
modulus, and gelation time) by simply varying the PEG molecular
weight (MW). We also demonstrated that the composite hydrogels
could support 3D encapsulation of human MSCs (hMSCs) with high
viability as well as tunable cell-hydrogel interactions by varying
the properties of hydrogels. The ability of fine-tuning hydrogel
properties paves the way for further optimization of cellular
responses to promote in situ tissue regeneration.
[0081] Materials and Methods
[0082] Hyaluronic acid sodium salt (HA) with molecular weight (MW)
of 2-3 million Daltons and chondroitin sulfate sodium salt (CS)
with average MW 10-30k Daltons were purchased from Carbosynth
Limited (Berkshire, UK). 1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), and polyethylene glycol diacrylate (PEGDA) with
MW of 700, 3400, and 8000 were purchased from Alfa Aesar (Ward
Hill, Mass.). PEGDA700 was passed through a short column containing
basic alumina to remove the inhibitor before use. PEGDA3400 and
PEGDA8000 were precipitated in diethyl ether twice to remove the
inhibitor before use. PEG divinylsulfone, (MW 3500 Daltons) and 4
arm PEG vinylsulfone (MW 20000 Daltons), were purchased from JenKem
Technology USA Inc. N-hyroxysuccinimide (NHS), cystamine
dihydrochloride, 5, 5'-dithiobis (2-nitrobenzoic acid) (Ellman's
Reagent), DL-Dithiotreitol (DTT), and 2-(N-morpholino)
ethanesulfonic acid (MES) were purchased from Sigma-Aldrich (St.
Louis, Mo.). 10.times. Phosphate buffer saline (PBS) was purchased
from Fisher Bioreagents (Pittsburgh, Pa.). Penicillin-Streptomycin
(Pen Strep), ethidium homodimer-1 (ethD-1), and calcein AM were
purchased from Life Technologies (Carlsbad, Calif.).
[0083] Synthesis of HA-SH and CS--SH
[0084] Carboxyl groups in HA and CS were functionalized to thiol
groups by a simple two-step reaction scheme which is depicted in
FIG. 7. In a typical procedure, HA (1 g, 2.5 mmol) was dissolved in
100 mL MES buffer (0.1 M MES, pH 6.0) in a 45.degree. C. oil bath
and allowed to dissolve overnight. EDC (2.4 g, 12.5 mmol) and NHS
(3.9 g, 34 mmol) were added and allowed to react for 2 h. Then
cystamine dihydrochloride (5.65 g, 25 mmol) was added to the
mixture and allowed to react overnight while stirring. The reaction
mixture was exhaustively dialyzed (MWCO of 12-14,000) against
distilled 0.1M NaCl for 60 h, 25% ethanol for 12 h and distilled
(DI) water for 12 h. After dialysis, the product, HA-conjugated
cystamine (HA-S--S--NH.sub.2), was lyophilized and kept at in
-20.degree. C. HA-conjugated cystamine (0.25 g) was dissolved in
PBS with a concentration of 5 mg/mL Then DTT (0.75 g, 4.8 mmol) was
added to the flask and pH was adjusted to 7.4 with 1 M NaOH. NaCl
was added to produce a 5% w/v solution after 20 h and the modified
HA was precipitated in 10-fold ethanol three times. The
precipitation was dissolved in H.sub.2O at a concentration of
approximately 5 mg/mL and the purified product was freeze-dried and
kept at -80.degree. C. freezer.
[0085] In a typical procedure, CS (1 g, 2 mmol) was dissolved in 40
mL MES buffer (0.1M MES, pH 6.0). EDC (3.086 g, 16.2 mmol) and NHS
(2.280 g, 19.8 mmol) were added to the flask and allowed to react
for 2 h. Following the 2 h activation step, the pH was raised to
7.2 using 1 M NaOH. Cystamine dihydrochloride (4.460 g, 20 mmol)
was subsequently added to the solution and allowed to react for
overnight. The CS-conjugated cystamine (CS--S--S--NH.sub.2) was
exhaustively dialyzed (MWCO of 12-14,000) against distilled 0.1M
NaCl for 60 h, 25% ethanol for 12 h and DI water for 12 h and then
was lyophilized. The CS--S--S--NH.sub.2 was reduced using DTT as
the same procedure of reduction of HA. The structure of
functionalized-HA or CS were confirmed .sup.1H NMR spectroscopy
(D.sub.2O, Bruker ARX 400 MHz) and the degree of thiolation was
also confirmed with an Ellman's assay using L-cysteine as the
standard.
[0086] Characterization of HA-SH and CS--SH
[0087] The structures of HA and CS-conjugated cystamines were
confirmed by proton nuclear magnetic resonance (.sup.1H NMR)
spectroscopy, with the degree of modification (DS) estimated from
integration of methylene protons relative to the N-acetyl methyl
protons in FIG. 8. By varying the molar ratios of HA/EDC/NHS,
variable DS values (4.1-43.1%) were obtained (Table 1, which shows
the effects of EDC concentration on degree of substitution (DS) of
--NH.sub.2.). For example, molar ratios of 1:5:13.5 and 1:10:13.5
yielded HA-conjugated cystamines with DS of 27% and 43.1%,
respectively. Therefore, CS/EDC/NHS molar ratio of 1:10:13.5 was
selected for CS modification.
TABLE-US-00001 TABLE 1 The effects of EDC concentration on degree
of substitution (DS) of --NH2. Reaction condition DS % of
--NH.sub.2 HA/EDC = 1:2 4.1 HA/EDC = 1:5 27 HA/EDC = 1:10 43.5
Subsequently, reduction of HA and CS-conjugated cystamines by
dithiotreitol (DTT) resulted in free thiol groups. The two-pot
reaction allows for efficient control of DTT reduction reactions
and minimizes the oxidation of thiols resulting in the disulfide
formation and the insolubility of HA or CS. DS of both amine and
thiol groups were controlled by simply adapting the pH of reaction
mixture or the amount of DTT. The results of DS determined by the
Ellman's assay and .sup.1H NMR were consistent and listed in Table
2, which shows the effects of DTT concentration and pH on DS of
--SH.
TABLE-US-00002 TABLE 2 The effects of DTT concentration and pH on
DS of --SH. DS % of --SH DS % of --SH Samples Reaction condition
(NMR) Ellman'sassay HA-SH 8-fold DTT 38.3 5.2 HA-SH 15-fold DTT
18.0 15.3 CS-SH PBS buffer 38.8 34.1 CS-SH pH = 7.2 25.5 23.5 CS-SH
pH = 8 18.5 17.7
[0088] .sup.1H NMR spectra (FIG. 9) showed the decrease of proton
signals from the cystamines disulfide pendants. Peak at d is
attributed to proton signals of methylenes
(--CH.sub.2CH.sub.2NH.sub.2). Peaks at a, b, a' and b' correspond
to resonances of the two side chain methylenes
(--CH.sub.2CH.sub.2SH). By altering the pH of reaction mixture, the
thiol group DS of CS--SH could be controlled from 17.7% to 36.0%
(Table 2). The pH at 7.2 was selected to offer reactive thiolate
and avoid the possible degradation of thiolated HA. By altering the
amount of DTT, the thiol group DS of HA-SH with 5.2% and 15.3%
respectively were obtained (Table 2). For the remaining studies,
HA-SH with a thiol group DS of 5.3% was employed because lower
substituted HA hydrogels have been shown to display higher cellular
bioactivity [23]. CS--SH with a thiol group DS of 23.5% was
employed. Moreover, the residual --NH.sub.2 groups allow for
additional functionalization of the hydrogels if desired.
[0089] The Formation of Hydrogels Using Thiol-Ene Chemistry
[0090] Crosslinking of HA-SH and CS--SH mixtures was achieved using
PEGDA as shown in FIG. 6. To study the effects of chain length of
cross-linker, the MW of PEGDA was altered to distribute either 700
Da, 3400 Da, or 8000 Da within hydrogel while the molar ratio of
thiol groups to acrylate groups was kept at 1.07 and all other
conditions were fixed (Table 3, which shows the composition of HCP
hydrogels). Hydrogels were formed by simple mixing of HA-SH,
CS--SH, and PEGDA in PBS at 37.degree. C. Specifically, 1% HA-SH
(199 .mu.L) and 5% CS--SH (217 .mu.L) were mixed in the
microcentrifuge tube. Either 5% PEGDA 700 (26.8 .mu.L), 20% PEGDA
3400 (33.8 .mu.L), or 33% PEGDA 8000 (46 .mu.L), was added to the
microcentrifuge tube to create HA/CS/PEGDA 700 (HCP700),
HA/CS/PEGDA 3400 (HCP3400), and HA/CS/PEGDA8000(HCP8000)
respectively.
TABLE-US-00003 TABLE 3 The composition of hydrogel MW of Polymer
m:m:HA-SH PEGDA percentage Hydrogel Type CS-aSH SH/-ene DS of HA DS
of CS (Da) (w/v %) HCP700L 1:5.1 1.07 5.1 .+-. 0.2 23.8 .+-. 1.8
700 3.2 HCP3400L 1:5.1 1.07 5.1 .+-. 0.2 23.8 .+-. 1.8 3400 4.4
HCP8000L 1:5.1 1.07 5.1 .+-. 0.2 23.8 .+-. 1.8 8000 6.2 HCP700
1:5.1 1.07 11.2 .+-. 0.9 32.9 .+-. 1.9 700 3.2 HCP3400 1:5.1 1.07
11.2 .+-. 0.9 32.5 .+-. 1.9 3400 4.8 HCP8000 1:5.1 1.07 11.2 .+-.
0.9 32.5 .+-. 1.9 8000 7.4
[0091] Cryo Scanning Electron Microscope (Cryo-SEM)
[0092] Hydrogel samples were imaged using an FEI NOVA nanoSEM field
emission scanning electron microscope (FEI Company, Hillsboro
Oreg.) using ET (Everhart-Thornley) dectector or the
high-resolution thorough-the lens (TLD) detector operating at 5 kV
accelerating voltage, spot 3, .about.5.0 mm working distance and 30
mm aperture.
[0093] The composite hydrogels exhibited interconnected porous
structures with micron-sized pores as shown in SEM micrographs
(FIG. 11). Generally, HCP700 hydrogels possessed larger pores than
HCP3400 and HCP8000 hydrogels presumably due to lower cross-linking
density of hydrogels.
[0094] Gelation Time
[0095] The time to form a gel (denoted as gelation time) is defined
as the time when the gel, in an inverted state, shows no fluidity
for 1 min[24] (FIG. 10). The experiment was performed in
triplicate. The gelation times decreased with increasing molecular
weight of PEGDA (p<0.05, n=3) (FIG. 12A). HCP700, HCP3400 and
HCP8000 hydrogels exhibited a gelation time of .about.30 min,
.about.18 min, and .about.16 min respectively.
[0096] Swelling Tests
[0097] For swelling tests, hydrogel samples (.about.0.35 mL) were
prepared as described above. The hydrogels were freeze-dried and
weighed (W.sub.d). Subsequently, 1 mL of PBS was applied on top of
the hydrogels and then the samples were incubated at 37.degree. C.
for 24 h to reach the swelling equilibrium. The excess PBS was then
aspirated away and the remaining saturated hydrogels were weighed
(W.sub.s). The experiments were performed in triplicate and the
degree of swelling of the hydrogels was expressed as
((W.sub.s-W.sub.d)/W.sub.d).times.100%. All data are presented as
mean.+-.standard deviation. The swelling ratio is another important
parameter for hydrogels, which is associated with hydrogel
mechanical properties including strength and flexibility.
[0098] All the composite hydrogels were highly swollen in water
resulting from the hydrophilicity of PEG, HA and CS molecules, and
the maintenance of cross-linked HA/CS/PEG networks. As shown in
FIG. 12B, the swelling ratio decreased significantly with the
increase of MW of PEGDA from 700 Da to 3400 Da (p<0.05, n=3).
Although not significant, a similar trend was also found while
increasing MW of PEGDA from 3400 Da to 8000 Da.
[0099] Drug Release Study
[0100] Tricomponent hydrogels (350 .mu.L) with different PEG
molecular weights (700, 3400, and 8000) were prepared in a syringe
with the end cut off. FITC-dextran (MW=70k, 20 .mu.g) was added to
each hydrogel precursor solution. The syringe was then placed in a
37.degree. C. incubator. The hydrogels were equilibrated for 2 h at
37.degree. C. After the two hours the hydrogel was then dispensed
into cell culture inserts (12 mm diameter with 3 .mu.m pore size
(Corning Incorporated, USA) in a 12 well plate. The hydrogels were
then submerged with PBS and the well plate was placed into a
37.degree. C. water bath. At specified time intervals, 1 mL of the
solution from individual wells was withdrawn and replaced with
pre-heated water. The amounts of released FITC-dextran were
determined by fluorescence measurements (excitation at 485 nm,
emission at 528 nm).
[0101] FIG. 12C shows the percent cumulative release profiles of
FITC-dextran from different composite hydrogels as a function of
time at 37.degree. C. After 58 hours, the FITC-dextran encapsulated
in HCP 700, HCP3400, and HCP8000 hydrogels resulted in about 84.3%,
77.6%, and 72.2% cumulative release of total FITC-dextran,
respectively. The release of FITC-dextran from HCP hydrogels was
mainly controlled by the diffusion. The release rate correlated
well with the hydrogel pore size as a functional of PEGDA molecular
weight. Increase of PEGDA chain length generates greater
crosslinking density resulting in smaller pore size and slower
release of FITC-dextran. This observation was consistent with our
swelling ratio results.
[0102] Rheological Characterization
[0103] Rheological experiments were carried out with a new
Discovery Series Hybrid Rheometer (DHR)-3 (TA) using parallel plate
(20 mm diameter, 0.degree.) in the oscillatory mode. Oscillatory
time, frequency, and strain sweeps were performed at 37.degree. C.,
and the storage (G') and loss (G'') moduli were recorded. 450 .mu.L
of gel precursor solution was mixed by vortexing at room
temperature for 15 s before loading on to the rheometer. Hydrogels
were cast between the lower Peltier plate (preheated at 37.degree.
C.) and upper parallel plate. The 20 mm parallel plate geometry was
set to a gap of 1000 .mu.m. Each hydrogel sample was used for only
one test. Strain sweeps and frequency sweeps were performed in
duplicate and the data represents the average of the two tests. 1%
strain and a frequency of 1 Hz were used for the time sweeps, with
the same 20 mm parallel plate for 4800 S. Time sweep tests were
performed in triplicate and the data represents the average of the
three tests with corresponding standard deviation.
[0104] The composite hydrogels showed tunable rheological and
mechanical properties by varying the PEGDA chain length. A strain
sweep from 0.1% to 10% strain was conducted at a frequency of 1.0
Hz (chosen arbitrarily) on a formed gel. FIG. 13A showed constant
storage modulus G' values as strain is varied up to 10% strain,
indicating that strains in the range 0.1% to 10% was in the
linear-viscoelastic regime (LVE)[25]. Consequently, a strain of 1%
was selected for the subsequent frequency sweep tests. At low
frequencies from 0.1 to 10 rad/s, G' does not change (FIG. 13B)
indicating the solid-like nature of the gel. As a result, a
frequency of 1 Hz was selected for future tests based on a
favorable torque signal at that frequency. A time sweep was
performed under the condition that the strain was small enough to
be in the LVE and the frequency was a value under which the
instrument provides a clear and favorable torque signal. Therefore,
1% strain, 1 Hz, and 37.degree. C. were determined to be the
appropriate conditions for the hydrogel time sweep tests.
HA/CS/PEGDA formed a gel and reached stability in .about.45 min
(FIG. 13C). However, the hydrogels formed rapidly and the crossover
of G' and G'' was not observed (G'' was too low to measure for the
extent of the tests). As noted in FIG. 13C, the gels have a
solid-like viscoelastic behavior. Extensive rheology testing showed
that by increasing the MW of the PEGDA, a significant increase in
the hydrogel storage modulus (p<0.05, FIG. 13D) was observed,
which is in good agreement with the conclusion derived from the SEM
data. Accordingly, HCP700 hydrogels were the weakest with a G'
value of 410.+-.30 Pa, whereas HCP8000 hydrogels were the strongest
with a G' value of 1810.+-.106 Pa. Storage modulus of HCP3400
hydrogels was 1300.+-.190 Pa, approximately triple that of HCP700
hydrogels.
[0105] 3D Cell Encapsulation and Culture
[0106] hMSCs were obtained from Lonza. Cells were cultured in
growth medium containing DMEM (Life technologies, catalog
#11885084) supplemented with 10% fetal bovine serum (FBS, Atlanta
Biologicals), 1% glutamine (Life technologies, catalog #25030081),
and 1% penicillin/streptomycin (Life technologies, catalog
#15140122) at 37.degree. C. with 5% CO.sub.2. For the cell
encapsulation in the hydrogels, cell pellets were obtained by
centrifugation, followed by a brief resuspension with hydrogel
solution through pipetting up and down gently to ensure a
homogeneous distribution. Then cell suspension with the density of
20,000 cells/25 .mu.L was transferred to an inverted pre-cut 1 mL
syringe and incubated at 37.degree. C. for 45 min. (FIG. 14).
Subsequently, hydrogels were injected to 24 well tissue culture
plate and incubated in growth medium. The medium was changed every
other day thereafter.
[0107] Cell Viability: Live/Dead Staining
[0108] The viability of the cells encapsulated in hydrogels after 1
and 5 days' culture was investigated by the live/dead viability
assay (Life technologies, catalog# L3224) according to the
manufacturer's instructions. Media were removed from all the
samples, washed twice with sterile PBS and stained with calcein-AM
and ethidium homodimer-1 solution. Samples in staining solution
were incubated at room temperature for 30 min. Imaging was
performed with a confocal microscope (Nikon AIR).
[0109] Mesenchymal stem cells (MSCs) offer a promising cell source
for musculoskeletal regenerative engineering due to their ease
availability, high expansion capacity, and multipotency. Thus,
hMSCs were encapsulated in the composite hydrogels and
characterized for cell viability and cellular responses. Confocal
images using a live/dead assay demonstrated that all composite
hydrogels supported progressive cellular growth with high viability
(>95%) during cell culture (FIG. 15 and FIG. 16). Cells
encapsulated within hydrogels displayed an overall spherical
morphology, which is in line with previous literature reports for
3D hydrogel cell culture[26].
[0110] Actin Immunofluorescence
[0111] Actin immunostaining was performed with the actin
cytoskeleton and focal adhesion staining kit (Millipore). Briefly,
cell/hydrogels were fixed in 4% paraformaldehyde and blocked with
1% BSA in PBS. Then samples were stained with TRITC-conjugated for
phalloidin F-actin and with DAPI for nuclei. Imaging was performed
with a confocal microscope (Nikon A1R).
[0112] Interestingly, hMSCs responded to differences in the
mechanical properties of hydrogels as evidenced from the changes in
actin cytoskeleton. In specific, hMSCs encapsulated in HCP700
hydrogels showed less defined actin fibers as compared to those in
HCP3400 and HCP8000 hydrogels (FIG. 17). Furthermore, cortical
actin protrusions into the surrounding hydrogel matrix were only
observed with hMSCs encapsulated in HCP3400 and HCP8000
hydrogels.
[0113] Focal Adhesion Kinase (FAK)
[0114] hMSC cells were incubated with a cell permeable focal
adhesion kinase (FAK) phosphorylation biosensor and were washed
gently with PBS (3.times.1 mL). After FAK biosensors were
successfully delivered into hMSCs, they were encapsulated in HCP
hydrogels. The dynamic monitoring of focal adhesion kinase (FAK)
activity was conducted to examine cell-hydrogel interactions using
fluorescence lifetime imaging microscopy (FLIM)[27].
[0115] The dynamic monitoring of FAK activity using FLIM confirmed
a similar finding where stiffened hydrogels exhibited increased FAK
phosphorylation of hMSCs (FIG. 18). It is important to note that
cells in different tissues are tuned to the specific mechanical
environments in which they reside [28].
[0116] 3D Cell Adhesion and Culture
[0117] The prepared HCP hydrogels were freeze-dried, sterilized for
20 min under UV light, and pre-incubated in the growth medium
containing DMEM (Life technologies, catalog #11885084) which was
supplemented with 10% fetal bovine serum (FBS, Atlanta
Biologicals), 1% glutamine (Life technologies, catalog #25030081),
and 1% penicillin/streptomycin (Life technologies, catalog
#15140122). hMSCs were seeded on the surface of the lyophilized
hydrogel scaffolds with a density of 20,000 cells/per scaffold and
were pre-incubated at 37.degree. C. with 5% CO.sub.2 for 1 h.
Subsequently, the lyophilized hydrogel scaffolds seeded with hMSCs
were cultured in 500 .mu.L growth medium at 37.degree. C. with 5%
CO.sub.2 for 1 day.
[0118] SEM Characterization
[0119] Cell seeded hydrogel scaffolds were dehydrated sequentially
with 50, 70, 80, 90, 95, and 100% ethanol for 10 minutes each and
then were freezer dried. The samples were then cut to expose the
cross-sectional area as well as the surface, and then sputter
coated with platinum for 60 seconds. The hydrogels were examined on
a scanning electron microscope (Nova NanoSEM) at 3.0 kV.
[0120] The lyophilized HCP hydrogel scaffolds were shown to have
porous structures (FIG. 19A, C) and to be able to support cell
growth on the surface of scaffolds (FIG. 19A, B). Pore structure
was roughly 40 to 50 .mu.m in diameter.
Example 2. HA/CS/Poly (Ethylene Glycol) Divinyl Sulfone (PEGVS)
Composite Hydrogel as Primary Chondrocytes Niches
[0121] The Formation Hydrogels using Michael-type Addition
Chemistry
[0122] In all experiments, the molar ratio --SH/-ene concentration
was held constant. Specifically, 1% HA-SH (192 .mu.L), 5% CS--SH
(199.2 .mu.L), and poly (ethylene glycol) divinyl sulfone (PEGVS)
3400 (52.8 .mu.L) were mixed at 37.degree. C. in the
microcentrifuge tube to create HA/CS/PEGVS 3400 (HCPV3400).
[0123] Rheological Characterization
[0124] Rheological experiments were carried out with a new
Discovery Series Hybrid Rheometer (DHR)-3 (TA) using parallel plate
(20 mm diameter, 0.degree.) in the oscillatory mode. Oscillatory
time, frequency, and strain sweeps were performed at 37.degree. C.,
and the storage (G') and loss (G'') moduli were recorded. 450 .mu.L
of gel precursor solution was mixed by vortexing at room
temperature for 15 s before loading on to the rheometer. Hydrogels
were cast between the lower Peltier plate (preheated at 37.degree.
C.) and upper parallel plate. The 20 mm parallel plate geometry was
set to a gap of 1000 .mu.m. Each hydrogel sample was used for only
one test. Time sweep tests were performed under 1% strain, 1 Hz,
and 37.degree. C. conditions (n=4). The data represents the average
of the three tests with corresponding standard deviation.
[0125] Due to more reactive vinyl sulfone moiety, PEGVS
demonstrates significantly shorter gelation time and higher storage
modulus compared to PEGDA (FIG. 20).
[0126] Chondrocytes Isolation
[0127] Chondrocyte cultures were prepared from pig articular
cartilage[29]. Shavings of cartilage were removed from the outside
of the articular cartilage, such that contamination with bone cells
or other connective tissue cells could be avoided. The pig
cartilage was finely chopped and the chondrocytes were released
from their extracellular matrix by sequential digestion at
37.degree. C. with collagenase. Cells obtained from the collagenase
digests were pooled and passed through a sterile 60 fan aperture
nylon screen (Nitex) to remove any undigested cartilage
fragments.
[0128] 3D Cell Encapsulation and Culture
[0129] Primary chondrocytes were cultured in growth medium
containing DMEM (Life technologies, catalog #11885084) supplemented
with 20% fetal bovine serum (FBS, Atlanta Biologicals), and 1%
penicillin/streptomycin (Life technologies, catalog #15140122) at
37.degree. C. with 5% CO.sub.2. For the cell encapsulation in the
hydrogels, cell pellets were obtained by centrifugation, followed
by a brief resuspension with hydrogel solution through pipetting up
and down gently to ensure a homogeneous distribution. Then cell
suspension with the density of 20,000 cells/25 .mu.L was
transferred to an inverted pre-cut 1 mL syringe and incubated at
37.degree. C. for 45 min. (FIG. 14). Subsequently, hydrogels were
injected to a 24 well tissue culture plate and incubated in growth
medium. The medium was changed every other day thereafter.
[0130] Cell Viability and Immunofluorescence
[0131] Actin immunostaining was performed with the actin
cytoskeleton and focal adhesion staining kit (Millipore). Briefly,
cell/hydrogels were fixed in 4% paraformaldehyde and blocked with
1% BSA in PBS. Then samples were stained with TRITC-conjugated
phalloidin for F-actin, with aggrecan antibody (H-300) (Santa Cruz
Biotech) for aggrecan, and with DAPI for nuclei. Imaging was
performed with a confocal microscope (Nikon AIR).
[0132] Confocal images using a live/dead assay demonstrated that
all composite hydrogels supported cellular growth with high
viability after a 21-day culture (FIG. 21A). Primary chondrocytes
are easy to de-differentiate after passages. Here we show that our
hydrogel can maintain chondrocytesm morphology after 21 days
indicated by the staining of aggrecan (FIG. 21B).
Example 3: HA/CS/4-Arm Poly (Ethylene Glycol) Divinyl Sulfone
(4PEGVS) Composite Hydrogel as hMSC Niches
[0133] The Formation Hydrogels using Michael-type Addition
Chemistry
[0134] In all experiments, the molar ratio --SH/-ene concentration
was held constant. Specifically, 1% HA-SH (140 .mu.L), 5% CS--SH
(150 .mu.L), and 25% 4-arm PEG) divinyl sulfone (4PEGVS) 20k (113
.mu.L) were mixed in the 1.5 mL centrifuge tube to create
HA/CS/4PEGVS 3400 (HCPV3400).
[0135] Rheological Characterization
[0136] Rheological experiments were carried out with a new
Discovery Series Hybrid Rheometer (DHR)-3 (TA) using parallel plate
(20 mm diameter, 0.degree.) in the oscillatory mode. Oscillatory
time, frequency, and strain sweeps were performed at 37.degree. C.,
and the storage (G') and loss (G'') moduli were recorded. 450 .mu.L
of gel precursor solution was mixed by vortexing at room
temperature for 15 s before loading on to the rheometer. Hydrogels
were cast between the lower Peltier plate (preheated at 37.degree.
C.) and upper parallel plate. The 20 mm parallel plate geometry was
set to a gap of 1000 .mu.m. Each hydrogel sample was used for only
one test. Time sweep tests were performed under 1% strain, 1 Hz,
and 37.degree. C. conditions (n=4). The data represents the average
of the three tests with corresponding standard deviation.
[0137] Compared with 2-arm PEGVS, 4-arm PEGVS demonstrates
significantly shorter gelation time and higher storage modulus
compared to PEGDA (FIG. 22).
[0138] 3D Cell Encapsulation and Culture
[0139] hMSCs were cultured in growth medium at 37.degree. C. with
5% CO.sub.2. For the cell encapsulation in the hydrogels, cell
pellets were obtained by centrifugation, followed by a brief
resuspension with hydrogel solution through pipetting up and down
gently to ensure a homogeneous distribution. Then the cell
suspension with a density of 20,000 cells/25 .mu.L was transferred
to an inverted pre-cut 1 mL syringe and incubated at 37.degree. C.
for 30 min. (FIG. 14). Subsequently, hydrogels were injected to a
24 well tissue culture plate and incubated in growth medium. The
medium was changed every other day thereafter.
[0140] Cell Viability and Actin Immunofluorescence
[0141] Confocal images using a live/dead assay demonstrated that
all composite hydrogels supported cellular growth with high
viability (>95%) after 21 days (FIG. 22A). Cells encapsulated
within hydrogels displayed an overall spherical morphology (FIG.
22B), which is in line with previous literature reports for 3D
hydrogel cell culture[26].
Example 4: Bio-inspired Composite Hydrogels for Osteochondral
Regenerative Engineering
[0142] Treatment of osteochondral defects encompassing injury or
degeneration to both the articular cartilage as well as the
underlying subchondral bone presents a significant medical
challenge. Current treatment options including autografts and
allografts suffer from limited availability and risk of
immunogenicity, respectively. The long term goal of this work is to
develop an integrated scaffold system for treatment of
osteochondral defects via in situ regeneration of bone, cartilage
and the bone-cartilage interface (FIG. 24). Hydrogels provide an
attractive biomaterial platform for regeneration of cartilage. In
the present disclosure, we have developed a novel composite
hydrogel that includes thiolated HA and CS cross-linked with PEG to
mimic cartilage. The bone mimetic is based on
PLGA/nano-hydroxyapatite composite scaffolds[30].
[0143] Here, integration between hydrogels and PLGA 3D scaffolds
was achieved using a novel multifunctional HA. HA was chemically
functionalized with both --SH and --NH.sub.2 groups to form two
functional arms: one arm to covalently bond to a cross-linker to
form hydrogels and the second arm bonding to the PLGA 3D scaffold
surface. This multifunctional HA provides a biologically active and
mechanically functional bridge with scaffolds.
[0144] PLGA 3D Scaffold Preparation
[0145] Poly (lactide-co-glycolide) (PLGA) 85:15 was purchased from
Lakeshore Biomaterials. PLGA microspheres were obtained using the
emulsion-solvent evaporation method. PLGA 3D scaffolds were
fabricated by heat sintering at an optimized condition[31].
[0146] PLGA 3D Scaffold-Hydrogel Integration and SEM
Characterization
[0147] The HA-SH, CS--SH and PEGDA solution (PBS) was placed onto
PLGA 3D scaffold surface for 45 min at 37.degree. C. PLGA 3D
scaffold-hydrogel integration was lyophilized and then sputter
coated with platinum for 60 seconds. The integration were examined
on a scanning electron microscope (Nova NanoSEM) at 3.0 kV. SEM
images of PLGA 3D scaffolds-hydrogel integration was shown in FIG.
25.
[0148] Statistical Analysis.
[0149] Differences among groups were assessed by one-way ANOVA with
Bonferroni post hoc correction to identify statistical differences
among three treatments. A p-value of 0.05 was set as the criteria
for statistical significance. Graphs are annotated where values are
represented as *0.05.
[0150] Additional disclosure is found in Appendix-A, filed
herewith, entirety of which is incorporated herein by reference
into the present disclosure.
[0151] Those skilled in the art will recognize that numerous
modifications can be made to the specific implementations described
above. The implementations should not be limited to the particular
limitations described. Other implementations may be possible. In
addition, all references cited herein are indicative of the level
of skill in the art and are hereby incorporated by reference in
their entirety.
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