Patent Description:
Glycosaminoglycans (GAGs) are long linear (unbranched) polysaccharides consisting of repeating disaccharide (double sugar) units. Glycosaminoglycans have good biocompatibility. Hyaluronic acid (HA; conjugate base hyaluronate), also called hyaluronan, is an anionic, nonsulfated glycosaminoglycan. It is a linear polysaccharide that consists of alternating units of a repeating disaccharide, β-<NUM>,<NUM>-D-glucuronic acid-β-<NUM>,<NUM>-N -acetyl-D-glucosamine. It is abundant in cartilage and skin and plays a key structural role in the organization of the extracellular matrix as an organizing structure for the assembly of a proteoglycan. Viscous solutions of high molecular-weight HA and its derivatives are being used in therapy for promoting wound healing in various tissues, such as a surgical aid in eye and skin. However, these solution systems are limited in application, due to undesirable loss of material from the injection site and minimal control over important material properties (e.g., mechanics and degradation).

Injectable hydrogels may be surgically implanted to fit variable target sites in patients using minimally invasive methods, making them particularly attractive for clinical use. For these purposes, a variety of injectable HA-based hydrogels have been explored by numerous chemical crosslinking mechanisms including photo-crosslinking, dynamic-chemistry, physical-assembly and non-covalent interactions.

For example, in <CIT>, HA was functionalized with thiol groups and subjected to a one-step crosslinking protocol for an injectable HA hydrogel basing on oxidation-reduction reactions between thiols and disulfide bond. Unfortunately, these materials are inherently limited in that they typically exhibit low mechanical strength and may exhibit rapid degradation.

<NPL>) disclosed an injectable hyaluronic acid-dextran hydrogel, wherein hyaluronic acid (HA) and dextran were chemically modified and subsequently crosslinked via formation of hydrazone (acylhydrazone) bonds in phosphate buffer. The compressive moduli of the hydrogels were around or above <NUM> kPa.

Hydrogels formed through covalent means display great versatility with the allowed inclusion of controlled network degradation and mechanical strength. In <CIT>, a UV-crosslinked, stiff HA hydrogel was prepared by free-radical photopolymerization of methacrylates, where HA were functionalized with both <NUM>-aminoethyl methacrylate hydrochloride and L-lysine. However, as a result of the single covalent bonding structure, hydrogels are unable of self-healing, which limits them in their applications as an injectable material.

<NPL>) disclosed a secondary photo-crosslinking of injectable shear-thinning dock-and-lock hydrogel, wherein the hydrogel was stabilized through light-initiated radical polymerization of methacrylate functional groups to tune gel mechanics and erosion kinetics. By tuning material compositions, hydrogels with storage modulus (G') as high as ~ <NUM> kPa could be formed.

For more physically demanding applications, non-covalently and covalently crosslinked systems may be more appropriate to modulate the injectability, self-healing properties and the degradation and mechanical strength. For example, in <CIT>, an injectable dual-crosslinked hydrogel system was prepared from amino-/vinyl- HA and oxidized HA. The hydrogels first undergo dynamic Schiff-based crosslinking through amino- and aldehyde- groups, then a secondary covalent crosslinking occurs in situ via vinyl- to stabilize the network. The compression moduli were around <NUM>. 4kPa~<NUM>.

<NPL>) disclosed a MeHA hydrogel with compression moduli around <NUM>~<NUM> kPa. The final storage modulus would be even lower according to the relationship between compression modulus and storage modulus: E = <NUM> × (<NUM>+µ) G. Here, E is Young's modulus. µ is Poisson's ratio, which is from <NUM> to <NUM>. G is shear modulus of elasticity.

In particular, a disadvantage with the Schiff-based reaction is exceedingly fast gelation, which may result in premature crosslinking and delivery failure. For ease of clinical application, the hydrogel must therefore undergo crosslinking with gentle reaction kinetics to prevent premature crosslinking and delivery failure.

There was thus the problem of providing a novel, mild HA-dextran hydrogel system which can self-heal immediately after shear induced flow, is cytocompatible, and can be stabilized through light-initiated radical polymerization of methacrylate functional groups to tune gel mechanics and degradation kinetics. Further, there was the problem of providing a process for the production of such hydrogels.

Stiffness has been regarded as a key metric for how the matrix resists cellular traction forces to regulate stem cell fate. For example, stiff substrates with moduli in the range of <NUM>-<NUM> kPa promote stem cell differentiation into osteoblasts. On the other hand, the phenotype of stem cell can be better maintained when they are cultured on soft substrates with a modulus of <NUM> kPa, as compared to those with a modulus of <NUM> kPa. From soft fat to stiff articular cartilage and bone, the moduli of these tissue are from <NUM> kPa to <NUM> kPa (see, <NPL>;<NPL>; and <NPL>). Therefore, a key goal is to achieve high moduli for hydrogel produced from natural materials thus with good biocompatibility and good tissue regeneration.

To address these inherent limitations of current injectable hydrogel systems, a novel, mild hydrogel system was developed by using mild dual-crosslinking conditions. To accomplish this, according to the present invention, a glycosaminoglycan such as HA is first functionalized with methacrylate and hydrazide groups, and a second-polysaccharide such as dextran is oxidized to obtain aldehyde groups. The mixture of functionalized glycosaminoglycan and second-polysaccharide-aldehyde first undergo gentle dynamic covalent acylhydrazone bond crosslinking through hydrazide and aldehyde groups to obtain a dynamic acylhydrazone bond cross-linked hydrogel. Acylhydrazone bond formation is an efficient, biocompatible chemistry, often used for bioconjugation, but also can be tuned to be dynamically covalent, depending on the chemical structures. See<NPL> for more description on acylhydrazone bond. This mechanism permits them to possess desirable properties like shear-thinning and rapidly self-healing. After injection of the dynamic acylhydrazone bond cross-linked hydrogel into a target site, a secondary covalent crosslinking occurs in situ via photopolymerization of methacrylates to stabilize the network and modulate mechanics to obtain the dual-crosslinked hydrogel.

The properties of the dual-crosslinked hydrogels permit them to be used as injectable and photo-stabilizing cell carriers. Cells such as stem cells can be homogenously encapsulated within the hydrogels under constant conditions by, for example, simply resuspending cells with for example one glycosaminoglycan component dissolved in growth media and subsequently mixing with the second polysaccharide component such as dextran-aldehyde. The dual-crosslinked hydrogels have good cytocompatibility and viable cells may be remained above <NUM> % for <NUM> days.

Without wishing to be bound by theory, it is believed that the dual-crosslinking, i.e., acylhydrazone crosslinking and methacrylate covalent crosslinking makes the dual-crosslinked hydrogel of the invention possesses advantages such as improved mechanical properties. The dual-crosslinked hydrogel of the invention may have a final storage modulus up to <NUM> kPa or more. Apparently, there is a synergetic effect between acylhydrazone crosslinking and methacrylate covalent crosslinking as none of the acylhydrazone crosslinking and methacrylate covalent crosslinking alone may achieve such high modulus. Such high modulus is particularly important and valuable for the applications such as scaffold for cartilage in tissue engineering. Thus, the dual-crosslinked hydrogel of the invention may be applied in applications which requires a high final storage modulus.

Compared with conventional hydrogels with dynamic acylhydrazone-crosslinking only, the dual-crosslinked hydrogel systems exhibit a slow reduction in mass loss at all observed time points. Moreover, the final storage modulus from photopolymerization of the dual-crosslinked hydrogel significantly increases from <NUM> kPa to <NUM> kPa or more, which is very surprising for and beyond expectation of a person skilled in the art.

The invention provides a process to prepare a dual-crosslinked hydrogel, comprising the following steps,.

The dual-crosslinked hydrogel of the invention comprises a first dynamic acylhydrazone bond crosslinking between hydrazide groups of hydrazide groups modified glycosaminoglycan and aldehyde groups of a second-polysaccharide-aldehyde, and a secondary covalent crosslinking formed in situ via photopolymerization of the methacrylate groups modified on the glycosaminoglycan.

The glycosaminoglycan of the invention is water soluble. The glycosaminoglycan is selected from glycosaminoglycans that may be modified by with a methacrylate on a hydroxyl group and with a dihydrazide on a carboxyl group. The glycosaminoglycan is preferably selected from the group consisting of hyaluronic acid, chondroitin sulfate (e.g. chondroitin-<NUM>-sulfate, and chondroitin-<NUM>-sulfate), dermatan sulfate, heparan sulfate and heparin, especially hyaluronic acid. Unless otherwise specified, the term "hyaluronic acid" in the invention refers to hyaluronic acid or a salt thereof, such as sodium hyaluronate. The salt of hyaluronic acid refers to water-soluble salt of hyaluronic acid. Non-limiting examples may be selected from sodium hyaluronate, hyaluronic acid potassium salt, tetrabutylammonium hyaluronate (HA-TBA), etc..

The second-polysaccharide of the invention is a water-soluble polysaccharide. The second-polysaccharide comprises a <NUM>,<NUM>-linkage and/or a <NUM>,<NUM>-linkage. In addition, the second-polysaccharide comprises an ortho-hydroxyl on C2, C3 or C4 of the sugar ring of the polysaccharide. The second-polysaccharide does not include those with <NUM>,<NUM>-linkage only. The second-polysaccharide may be glucans such as β-<NUM>,<NUM>/<NUM>,<NUM>-glucan, α-<NUM>,<NUM>-glucan with α-<NUM>,<NUM>-branches, α-<NUM>,<NUM>- glucan, α -<NUM>,<NUM>/<NUM>,<NUM>-glucan, alginate and pectin, etc. Non-limiting examples of the glucan include dextran, laminarin (β-<NUM>,<NUM>- and β-<NUM>,<NUM>-glucan), water soluble cellulose derivatives, especially dextran.

The term "methacrylate" refers to hydrolyzable methacrylates. Non-limiting examples of methacrylate include methacrylic anhydride, <NUM>-aminoethyl methacrylate hydrochloride, glycidyl methacrylate.

The dihydrazide in the invention refers to a dihydrazide that may, after hydrazide group modification of the glycosaminoglycan, further react with an aldehyde group to form an acylhydrazone bond. The dihydrazide may be selected from water-soluble dihydrazides. Non-limiting examples may be selected from adipic dihydrazide, ethanedihydrazide, oxalyldihydrazide, dodecanedioic dihydrazide.

In some embodiments, the dual-crosslinked hydrogel is a dual-crosslinked hyaluronic acid-dextran hydrogel.

Methacrylate may react with the free hydroxyl groups at different positions such as C6 of the hyaluronic acid repeating unit, although hydroxyl group of C6 is typically the most reactive one.

In the invention, the term "second-polysaccharide with aldehyde groups" is used interchangeably with "second-polysaccharide-aldehyde". Both terms mean second-polysaccharide with aldehyde groups oxidized from hydroxyl groups on the original second-polysaccharide molecule. Similarly, the term "dextran with aldehyde groups" is used interchangeably with "dextran-aldehyde". Both terms mean dextran with aldehyde groups oxidized from hydroxyl groups on the original dextran molecule.

The term "degree of methacrylation" refers to:
(the mole of methacrylated hydroxyl groups / the total mole number of repeating units contained in the polymer) * <NUM>%.

See<NPL> for more description on methcarylation degree and the determination method thereof.

The degree of methacrylation may be <NUM>-<NUM>%, preferably <NUM>-<NUM>%, for example <NUM>-<NUM>%, <NUM>-<NUM>%,<NUM>-<NUM>%,<NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%.

The term "degree of hydrazide group modification" refers to:
(the mole of hydrazide group modified repeating units/ the mole of repeating units in the polymer) * <NUM>%.

See <NPL> for the determination method thereof.

The degree of hydrazide group modification may be <NUM>-<NUM>%, preferably <NUM>-<NUM>%, for example <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%.

When adipic dihydrazide (ADH) and dextran is used, acylhydrazone bonds are formed between the aldehyde groups of dextran-aldehyde (oxidized dextran or ODEX) and the acylhydrazide bonds of ADH in step <NUM>).

A person skilled in the art may control the oxidation degree of the second-polysaccharide-aldehyde, e.g. through reaction time and the amount of the oxidant. The degree of aldehyde modification in the second-polysaccharide such as dextran product may be determined by e.g. measuring the number of aldehyde groups in the polymer using t-butyl carbazate and trinitrobenzenesulfonic acid, see for example,<NPL>; or <NPL>.

The degree of aldehyde modification in the second-polysaccharide maybe <NUM>-<NUM>%, for example <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%, preferably <NUM>-<NUM>%.

In order to obtain optimized gelation, a person skilled in the art may adjust the concentration of the modified glycosaminoglycan and modified second-polysaccharide; preferably the concentration of modified glycosaminoglycan such as modified hyaluronic acid is <NUM>-3wt. %, preferably <NUM>~2wt. %, the concentration of second-polysaccharide-aldehyde such as dextran-aldehyde is <NUM>-8wt. %, preferably <NUM>~6wt. % based on the total weight of the reaction mixture in step <NUM>).

In some embodiments, the glycosaminoglycan, such as hyaluronic acid may have a molecular weight of <NUM>-<NUM> kDa, preferably <NUM>-<NUM> kDa.

In some embodiments, the second-polysaccharide, such as dextran may have a molecular weight of <NUM>-<NUM> kDa, such as <NUM>-<NUM> kDa,<NUM>~<NUM> kDa, preferably <NUM>-<NUM> kDa.

In step <NUM>), glycosaminoglycan such as hyaluronic acid is modified with methacrylate to obtain a methacrylated glycosaminoglycan, then further modified with hydrazide groups. Alternatively, glycosaminoglycan may be modified with methacrylate and hydrazide groups separately (i.e., not in the same molecule), then in step <NUM>) crosslinking both the methacrylated glycosaminoglycan and hydrazide modified glycosaminoglycan with the second-polysaccharide-aldehyde to form a dynamic acylhydrazone bond cross-linked hydrogel. In addition, in step <NUM>), the second-polysaccharide is oxidized to prepare a second-polysaccharide-aldehyde.

In step <NUM>), the modified glycosaminoglycan and second-polysaccharide -aldehyde is crosslinked via formation of acylhydrozone bonds, preferably in a phosphate buffer saline (PBS) or a cell culture medium.

In step <NUM>), the methacrylate functional groups of the dynamic acylhydrazone bond cross-linked hydrogel are polymerized. In some examples, the polymerization is a light-initiated radical polymerization.

The irradiation may be <NUM>-<NUM> irradiation, sunlight irradiation, etc., preferably <NUM> blue light. In some embodiments, the dynamic acylhydrazone bond cross-linked hydrogels were crosslinked by using <NUM> blue-light, at a power of <NUM>-<NUM> W, a light intensity of <NUM>~<NUM> mW/cm<NUM> for <NUM>-<NUM>.

The photoinitiator may be those conventional in the art, for example lithium phenyl-<NUM>, <NUM>, <NUM>-trimethylbenzoylphosphinate (LAP). In some embodiments, the photoinitiator may be dissolved in the aqueous solvent of step <NUM>).

The invention further provides a dual-crosslinked hydrogel prepared according to the above process of the invention.

The invention further provides a hydrogel precursor to prepare a dual-crosslinked hydrogel, wherein the hydrogel precursor is prepared by a method comprising the following steps:.

The hydrogel precursor is the dynamic acylhydrazone bond cross-linked hydrogel prepared in step <NUM>). It is injectable and may be used to prepare the dual-crosslinked hydrogel of the invention.

In use, the hydrogel precursor may optionally carry active ingredients like cells and be injected into the desired part of a subject. Then the hydrogel precursor may be photopolymerized by exposure to irradiation (e.g. <NUM> blue light) in situ, thereby a dual-crosslinked hydrogel may be prepared.

The invention further provides use of the dual-crosslinked hydrogel of the invention or prepared according to the process of the invention or the hydrogel precursor of the invention in tissue engineering, bioapplications such as drug delivery system, biosensors and soft tissue filling agents, etc., or as a carrier of at least one active ingredient such as cell. The cell includes stem cells. The cell carrier may have a high cell viability. Notably, stem cells can be homogenously encapsulated within the dual-crosslinked hydrogel and the viable cells may be remained above <NUM> % for <NUM> days. In use, the cells may be incorporated into the dual-crosslinked hydrogel by various ways. For example, the cells may be incorporated into the dual-crosslinked hydrogel by impregnating the dynamic acylhydrazone bond cross-linked hydrogel in a cell suspension; or preferably cells may be added into the reaction mixture before forming the dynamic acylhydrazone bond cross-linked hydrogel, for example cells may be mixed with the functionalized glycosaminoglycan and/or second-polysaccharide with aldehyde groups.

The dual-crosslinked hydrogel with cell may be used as tissue induced scaffold. The dual-crosslinked hydrogel without cell may be used as tissue engineering scaffold.

Chemical crosslinking can be tuned by the dynamic acylhydrazone crosslinking and secondary photo-irradiation, and the extent of chemical crosslinking can be tuned by adjusting irradiation exposure time. The gelation time and equivalent time can be controlled within <NUM>-<NUM> and <NUM>-<NUM> mins, respectively for the dynamic acylhydrazone bond cross-linked hydrogel. With secondary crosslinking, the moduli of the hydrogels can be controlled within <NUM> kPa~<NUM> kPa, or be controlled from <NUM> kPa, <NUM> kPa, <NUM> kPa, <NUM> kPa, <NUM> kPa, <NUM> kPa, <NUM> kPa, or <NUM> kPa, to <NUM> kPa, even up to <NUM> kPa, <NUM> kPa, <NUM> kPa, <NUM> kPa, <NUM> kPa, <NUM> kPa, <NUM> kPa, <NUM> kPa, or <NUM> kPa, by e.g. controlling the degree of degree of methacrylation, the degree of hydrazide group modification, the degree of aldehyde modification, the molecular weight of glycosaminoglycan and second-polysaccharide, the concentration of modified glycosaminoglycan such as hyaluronic acid, the concentration of second-polysaccharide-aldehyde, and the irradiation exposure time between for example <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> to <NUM> mins, <NUM> mins, <NUM> mins, <NUM> mins, or <NUM> mins, such as <NUM> to <NUM> mins, <NUM> to <NUM> mins, <NUM> to <NUM> mins, <NUM> to <NUM> mins, <NUM> to <NUM> mins.

In some embodiments, the chemical structure of dual-functionalized HA with dihydrazide- and methacrylate- modification may be schematically shown as formula I:
<CHM>.

In some embodiments, the molecular weight of sodium hyaluronate is <NUM>~<NUM> kDa, for example, <NUM>~<NUM> kDa, preferably <NUM>-<NUM> KDa. In one embodiment, the molecular weight of sodium hyaluronate is <NUM>-<NUM> KDa.

In some embodiments, the structure of dextran-aldehyde (oxidized dextran or ODEX) may be schematically shown as formula II:
<CHM>.

In some embodiments, the dual-crosslinked hydrogel according to the invention is prepared according to the method as follows,.

In some embodiments, the purification is done bydialyzing the macromer solution against DI H<NUM>O (MW cutoff <NUM>,<NUM>-<NUM>,<NUM>) for <NUM>-<NUM> days.

The purified methacrylated HA may be frozen at -<NUM> to -<NUM>, lyophilized, and stored at -<NUM> in powder form.

Preparation of Dihydrazide-HA (DHA-HA) or Dihydrazide-MeHA (DHA-MeHA) Dissolving sodium hyaluronate or MeHA in H<NUM>O at a concentration of <NUM>-<NUM>/mL; Adding a <NUM>-fold molar excess of adipic dihydrazide to this solution;.

In some embodiments, the purification is done by exhaustively dialyzing (MW cutoff <NUM>,<NUM>-<NUM>,<NUM>) against distilled H<NUM>O for <NUM>-<NUM> days.

The yield of product may typically be around <NUM>%.

In some embodiments, the purification is done by exhaustive dialysis against water for <NUM>-<NUM> days. Dry product of the dextran-aldehyde may be obtained by freeze drying.

Dual-crosslinked MeHA/DHA-HA/ODEX or DHA-MeHA/ODEX hydrogel formation Preparing MeHA/DHA-HA or DHA-MeHA solutions (ranging from <NUM>-<NUM> wt. %) in PBS (pH <NUM>) containing <NUM>-<NUM> wt. % lithium phenyl-<NUM>, <NUM>, <NUM>-trimethylbenzoylphosphinate (LAP) photoinitiator;.

In some embodiments, <NUM> solutions of ODEX (<NUM>-<NUM>% w/v in PBS (Invitrogen)) were pipetted into <NUM> DHA-HA or DHA-MeHA (<NUM>-<NUM>% w/v) contained in a cylindrical mold made from a truncated <NUM>-mL syringe. The mixture was vigorously stirred using the pipet tip and then put on a shaker for gelation which typically took about <NUM>-<NUM>. The resulting round hydrogel disks were exposed to <NUM> mW cm-<NUM> <NUM> blue light for <NUM>-<NUM> on each side. Disk shaped hydrogels (~<NUM> width, ~<NUM> height) were cored out of the gel slab and immersed and equilibrated with PBS for <NUM>-<NUM>.

Therefore, the present invention provides a novel, mild dual-crosslinked hydrogel which has advantageous properties like rapid self-healing (although worse than the hydrogel precursor of the invention), and good cytocompatibility. In addition, the hydrogel precursor, i.e., dynamic acylhydrazone bond cross-linked hydrogel is injectable and has the property of shear-thinning before irradiation; the hydrogel can self-heal immediately after shear induced flow and can be stabilized through light-initiated radical polymerization of methacrylate functional groups to tune gel mechanics and degradation kinetics.

Other advantages of the present invention would be apparent for a person skilled in the art upon reading the specification.

The invention is now described in detail by the following examples.

Sodium hyaluronate (Evonik Industries AG, <NUM>-<NUM> kDa) was dissolved at in deionized water (DI H<NUM>O) to prepare a <NUM> wt% solution. Methacrylic anhydride (MA; Sigma) was added dropwise (~<NUM> per g HA) with stirring at <NUM>. The stirring mixture was maintained at pH ~<NUM> by continuously adding <NUM> NaOH for <NUM>, followed by reaction overnight at <NUM> and further addition of MA (~<NUM> MA per g HA) with pH maintenance for ~<NUM>. The obtained macromer solution was dialyzed against DI H<NUM>O (MW cutoff <NUM>,<NUM>-<NUM>,<NUM>) for <NUM> d, frozen at -<NUM>, lyophilized, and stored at -<NUM> in powder form. The modification degree of MeHA was <NUM>% according to <NUM>H-NMR analysis.

MeHA prepared above was dissolved in H<NUM>O at a concentration of <NUM>/mL. To this solution a <NUM>-fold molar excess of adipic dihydrazide was added. The pH of the reaction mixture was adjusted to <NUM> with <NUM> NaOH/<NUM> HCl. <NUM>-Ethyl-<NUM>-[<NUM> (dimethylamino)propyl]-carbodiimide (EDC) (<NUM>, <NUM> mmol; Aldrich Chemical Co. ) and <NUM>-hydroxybenzotriazole (HOBt) (<NUM>, <NUM> mmol; Aldrich Chemical Co. ) was dissolved in dimethylsulfoxide (DMSO)/H<NUM>O (<NUM>:<NUM>, <NUM>). After mixing, the pH of the reaction was maintained at <NUM> by the addition of <NUM> NaOH and the reaction was allowed to proceed overnight. The pH was subsequently adjusted to <NUM> with <NUM> NaOH and the derivatized HA exhaustively was dialyzed against DI H<NUM>O (MW cutoff <NUM>,<NUM>-<NUM>,<NUM>) for <NUM> d, frozen at -<NUM>, lyophilized, and stored at -<NUM> in powder form. The dihydrazide modification degree of DHA-MeHA was <NUM>% according to <NUM>H-NMR analysis.

To functionalize dextran (molecular weight: <NUM> kDa) with aldehyde groups, <NUM> dextran was dissolved in water at a concentration of <NUM>/mL. An aqueous solution of sodium periodate (<NUM> dissolved in <NUM> water) was added dropwise, and the reaction mixture was stirred for <NUM> at room temperature in the dark. Ethylene glycol was then added to inactivate any unreacted periodate. The solution was purified by exhaustive dialysis against water for <NUM> days, and the dry product of the dextran-aldehyde was obtained by freeze drying. The modification degree of dextran-aldehyde was <NUM>% according to trinitrobenzene sulfonate assay.

A <NUM> wt. % DHA-MeHA solution was prepared by dissolving DHA-MeHA in PBS (pH <NUM>) containing <NUM> wt. % lithium phenyl-<NUM>, <NUM>, <NUM>-trimethylbenzoylphosphinate (LAP) photoinitiator (Tokyo Chemical Industry Co. <NUM> ODEX (<NUM>% w/v prepared by dissolving ODEX in PBS (Invitrogen)) solution was pipetted into <NUM> DHA-MeHA (<NUM>% w/v in PBS) solution contained in a cylindrical mold made from a truncated <NUM>-mL syringe. The mixture was vigorously stirred using the pipet tip and then put on a shaker for gelation which typically took about <NUM>. The resulting round hydrogel disk, which was a dynamic acylhydrazone bond cross-linked hydrogel, was exposed to <NUM> mW/cm<NUM> <NUM> blue light for <NUM> on each side. The disk shaped dual-crosslinked hydrogel (~<NUM> width, ~<NUM> height) was cored out of the gel slab and immersed and equilibrated with PBS for <NUM>. Multiple dual-crosslinked hydrogel samples were prepared in Example <NUM>. The storage modulus of the obtained hydrogel was about <NUM> kPa.

A <NUM> wt. % solution of DHA-MeHA prepared during Example <NUM> was prepared in PBS (pH <NUM>) containing <NUM> wt. % lithium phenyl-<NUM>, <NUM>, <NUM>-trimethylbenzoylphosphinate (LAP) photoinitiator. LAP was chosen due to its aqueous solubility and good cytocompatibility. The mixtures were pipetted between glass slides and exposed to <NUM> mW/cm<NUM> <NUM> blue light for <NUM> on each side. Disk shaped hydrogels (~<NUM> width, ~<NUM> height) were cored out of the gel slab.

The modulus of the photo-crosslinked DHA-MeHA hydrogel was <NUM> kPa. The determination method was the same as the method described below used for Example <NUM>.

MeHA and dihydrazide-MeHA (DHA-MeHA) was prepared according to the same method as Example <NUM>.

Dextran-aldehyde was prepared according to the same method as Example <NUM>.

Dual-crosslinked DHA-MeHA/ODEX hydrogel was prepared according to the same method as Example <NUM> except that the resulting dynamic acylhydrazone-crosslinked hydrogel was exposed to <NUM> mW/cm<NUM> <NUM>-<NUM> irradiation (comprising <NUM> blue light) instead of <NUM> blue light. The storage modulus of the obtained hydrogel was about <NUM> kPa. As the irradiation was not done under the optimum irradiation wavelength <NUM>, the storage modulus was lower than that in Example <NUM>.

Dextran-aldehyde was prepared according to the same method as Example <NUM> except that the aqueous solution of sodium periodate used <NUM> instead of <NUM> sodium periodate dissolved in <NUM> water. The modification degree of dextran-aldehyde was <NUM>% according to trinitrobenzene sulfonate assay.

A <NUM> wt. % DHA-MeHA solution was prepared in PBS (pH <NUM>) containing <NUM> wt. % lithium phenyl-<NUM>, <NUM>, <NUM>-trimethylbenzoylphosphinate (LAP) photoinitiator. <NUM> ODEX (<NUM>% w/v in PBS) solution was pipetted into <NUM> DHA-MeHA (<NUM>% w/v in PBS) solution contained in a cylindrical mold made from a truncated <NUM>-mL syringe. The mixture was vigorously stirred using the pipet tip and then put on a shaker for gelation which typically took about <NUM>. The resulting round hydrogel disk was exposed to <NUM> mW/cm <NUM>-<NUM> irradiation (comprising <NUM> blue light) for <NUM> on each side. The disk-shaped hydrogel (~<NUM> width, ~<NUM> height) was cored out of the gel slab and immersed and equilibrated with PBS for <NUM>. The storage modulus of the obtained hydrogel was about <NUM> kPa. As the irradiation was not done under the optimum irradiation wavelength <NUM>, the storage modulus was lower than that in Example <NUM>.

Methacrylated HA (MeHA) was prepared according to the same method as Example <NUM>.

Sodium hyaluronate (Evonik Industries AG, <NUM>-<NUM> kDa) was dissolved in H<NUM>O at a concentration of <NUM>/mL. To this solution a <NUM>-fold molar excess of adipic dihydrazide was added. The pH of the reaction mixture was adjusted to <NUM> with <NUM> NaOH/<NUM> HCl. <NUM>-Ethyl-<NUM>-[<NUM> (dimethylamino)propyl]-carbodiimide (EDC) (<NUM>, <NUM> mmol) and <NUM>-hydroxybenzotriazole (HOBt) (<NUM>, <NUM> mmol) was dissolved in dimethylsulfoxide (DMSO)/H<NUM>O (<NUM>:<NUM>, <NUM>). After mixing, the pH of the reaction was maintained at <NUM> by the addition of <NUM> NaOH and the reaction was allowed to proceed overnight. The pH was subsequently adjusted to <NUM> with <NUM> NaOH and the derivatized HA exhaustively was dialyzed against DI H<NUM>O (MW cutoff <NUM>,<NUM>-<NUM>,<NUM>) for <NUM> d, frozen at -<NUM>, lyophilized, and stored at -<NUM> in powder form.

Dynamic acylhydrazone bond cross-linked hydrogel was prepared by mixing reactive DHA-HA, MeHA and dextran-aldehyde polymer solutions containing <NUM> wt. % lithium phenyl-<NUM>, <NUM>, <NUM>-trimethylbenzoylphosphinate (LAP) photoinitiator. Specifically, <NUM> ODEX (<NUM>% w/v in PBS) solution was pipetted into <NUM> DHA-HA/MeHA (<NUM>:<NUM>, <NUM>% w/v in PBS, which was prepared by mixing <NUM>% w/v DHA-HA in PBS with <NUM>% w/v MeHA in PBS) solution containing <NUM> wt. % LAP photoinitiator contained in a cylindrical mold made from a truncated <NUM>-mL syringe. The mixture was vigorously stirred using the pipet tip and then put on a shaker for gelation which typically took about <NUM>. The resulting round hydrogel disk was exposed to <NUM> mW/cm <NUM>-<NUM> irradiation (comprising <NUM> blue light) for <NUM> on each side. The resulting round hydrogel disk was ejected from the cylindrical mold, and immersed and equilibrated with PBS for <NUM>. The storage modulus of the obtained hydrogel was about <NUM> kPa. As the irradiation was not done under the optimum irradiation wavelength <NUM>, the storage modulus was lower than that in Example <NUM>.

Dihydrazide-HA (DHA-HA) was prepared according to the same method as Example <NUM>.

Dynamic acylhydrazone bond cross-linked hydrogel was prepared by mixing reactive DHA-HA, MeHA and dextran-aldehyde polymer solutions containing <NUM> wt. % lithium phenyl-<NUM>, <NUM>, <NUM>-trimethylbenzoylphosphinate (LAP) photoinitiator. Specifically, <NUM> ODEX (<NUM>% w/v in PBS) solution was pipetted into <NUM> DHA-HA/MeHA (<NUM>:<NUM>, <NUM>% w/v in PBS, which was prepared by mixing <NUM>% w/v DHA-HA in PBS with <NUM>% w/v MeHA in PBS) solution containing <NUM> wt. % LAP photoinitiator contained in a cylindrical mold made from a truncated <NUM>-mL syringe. The mixture was vigorously stirred using the pipet tip and then put on a shaker for gelation which typically took about <NUM>. The resulting round hydrogel disk was exposed to <NUM> mW/cm <NUM>-<NUM> irradiation (comprising <NUM> blue light) for <NUM> on each side. The disk shaped dual-crosslinked hydrogel (~<NUM> width, ~<NUM> height) was cored out of the gel slab and immersed and equilibrated with PBS for <NUM>. The storage modulus of the obtained hydrogel was about <NUM> kPa. As the irradiation was not done under the optimum irradiation wavelength <NUM>, the storage modulus was lower than that in Example <NUM>.

The MeHA, DHA-MeHA, and ODEX samples prepared during Example <NUM> and DHA-HA sample prepared during Example <NUM> were analyzed by using NMR spectrometer. The results were shown in <FIG>.

The chemical structure of functional HA derivatives including MeHA, DHA-HA, DHA-MeHA and dextran-aldehyde was determined on a Bruker Advance III spectrometer using D<NUM>O as the solvent. Proton-NMR of the modified HA revealed distinct methylene (HA <NUM> and <NUM> ppm). The degree of methacrylate modification was determined from the relative integrations of the methacrylate (<NUM> each) to the HA backbone protons (<NUM>).

As shown in <FIG>, for the MeHA, the signals of vinyl protons clearly appeared at <NUM> and <NUM> ppm. According to the peak areas of <NUM> ppm and <NUM> ppm relative to the sugar ring of HA (<NUM>-<NUM> ppm, <NUM>) in the MeHA spectrum, the degree of methacrylation was about <NUM>%.

The chemical structure of DHA-HA was shown in <FIG>, the apparent degree of modification calculated by the ratio of the protons of adipic (<NUM>, CH<NUM>CH<NUM>, <NUM>-<NUM> ppm) to the sugar ring of HA (<NUM>-<NUM> ppm, <NUM>) was about <NUM>%.

In the spectrum of the DHA-MeHA, the vinyl protons (<NUM> ppm, <NUM> ppm) and adipic protons (<NUM>, CH<NUM>CH<NUM>, <NUM>-<NUM> ppm) were shown in <FIG>, where the degree of methacrylation was <NUM>%, and the degree of hydrazide groups modification was <NUM>%.

In the spectra of dextran-aldehyde, ODEX presents several additional peaks in the range of <NUM>-<NUM> ppm, which were assigned to the protons from the hemiacetal structures (<FIG>).

Furthermore, the rheology tests (moduli, shear-thinning and self-healing) as shown below and in <FIG> confirmed that the desired dual-crosslinking structure were formed in the dynamic acylhydrazone bond cross-linked hydrogel prepared during Example <NUM>.

<FIG> indicated that dynamic acylhydrazone bond were formed in the dynamic acylhydrazone bond cross-linked hydrogel prepared during Example <NUM>.

<FIG> indicated that when irradiated, the dihydrazide-MeHA (DHA-MeHA) prepared during Comparative Example <NUM> may form double-bond crosslinking due to the existence of double-bonds in the methacrylation in the modified HA.

<FIG> indicated that when irradiated, dual-crosslinking were formed in the dual-crosslinked HA-dextran hydrogel prepared in Example <NUM>.

The hydrogels prepared during Example <NUM> were analyzed according to the methods as follow. The other hydrogels prepared in the description were analyzed according to the same methods.

Moduli, shear-thinning and self-healing of HA-dextran hydrogels prepared during Example <NUM> including the dynamic acylhydrazone bond cross-linked hydrogel and the dual-crosslinked DHA-MeHA/ODEX hydrogel were assessed with shear rate sweeps and time sweeps using a DHR-<NUM> rheometer (TA Instruments, Delaware, United States) with a quartz plate connected to a blue light source. A plate geometry with a solvent trap, <NUM> diameter, and <NUM> gap distance was used. Dynamic acylhydrazone bond cross-linked HA-dextran hydrogels prepared during Example <NUM> were formed by homogenously mixing together HA/dextran components, and loaded onto the rheometer. Shear rate swept between <NUM> to <NUM>-<NUM> was tested at <NUM>. Time sweeps were performed for shear recovery experiments. Hydrogels were deformed using <NUM>-<NUM> and allowed to recover at <NUM>-<NUM> shear rate at <NUM>. To measure the response of rheological properties to photopolymerization, in situ polymerization was performed with in-situ dynamic crosslinking with <NUM> wavelength irradiation at <NUM> mW/cm<NUM> light intensity using a dental lamp attached to a light guide for different formulations for <NUM>-<NUM> via a light-curing stage during oscillatory time sweeps at a frequency of <NUM> and a strain of <NUM>%. Experiments were repeated for a minimum of three times, and representative data was presented.

As shown in <FIG>, the viscosity of the dynamic acylhydrazone bond cross-linked hydrogel continuously decreased with shear rate increased from <NUM>-<NUM> to <NUM>-<NUM>, which suggests that such hydrogels exhibit shear-thinning property suitable for easy injection. Moreover, these hydrogels were able to self-heal after shear injection. As shown in <FIG>, at <NUM>-<NUM> shear rate, the viscosity of the dynamic acylhydrazone bond cross-linked HA-dextran hydrogel abruptly decreased from <NUM> Pa·s to <NUM> Pa·s. In contrast, as the shear rate was jumped to <NUM>-<NUM>, the viscosity rapidly recovered to its original value. These values demonstrate that the dynamic acylhydrazone bond cross-linked HA-dextran hydrogels self-healed rapidly into hydrogels and the methacrylate functionality did not interfere with the shear-thinning and self-healing properties of the dynamic hydrogels.

The representative curves of storage (G') and loss (G") moduli of the hydrogels prepared during Example <NUM> and Comparative Example <NUM> were shown in <FIG>. The storage modulus (G') and the loss modulus (G") of the hydrozide-crosslinking DHA-MeHA/ODEX were firstly monitored with an oscillatory time sweep.

<FIG> shows the dynamic acylhydrazone bond crosslinking step during Example <NUM>, the injectable liquid-like property of the precursor (before dynamic acylhydrazone bond crosslinking) was shown from the larger G" than G'. After crosslinking of <NUM>, the injectable precursor was converted into a gel (dynamic acylhydrazone bond cross-linked HA-dextran hydrogel), where the G' was larger than G". Allowing further crosslinking, the G' of dynamic DHA-MeHA/ODEX hydrogels reached ~<NUM> Pa at about <NUM> of crosslinking. These values suggested that the dynamic acylhydrazone bond cross-linked DHA-MeHA/ODEX hydrogels were soft and weak, which were beneficial for injection.

<FIG> shows the formation of DHA-MeHA hydrogel prepared during Comparative Example <NUM>. Upon exposure to free-radical generating irradiation using a <NUM> blue-light, the precursor (DHA-MeHA without ODEX component) exhibit increased in both G' and G", and was rapidly converted into a hydrogel (< <NUM>), as reflected by the increase in G' over the G". Allowing crosslinking to proceed to completion, the G' of DHA-MeHA hydrogels reached as high as ~<NUM> kPa at about <NUM> of irradiation.

<FIG> shows the formation of dual-crosslinked DHA-MeHA hydrogel prepared during Example <NUM>. As shown in <FIG>, the DHA-MeHA/ODEX hydrogels were firstly crosslinked by dynamic hydrazone and subsequently strengthened via chemical crosslinking. The dual-crosslinked DHA-MeHA/ODEX hydrogels resulted in a more rigid viscoelastic solid. The G' of the dual-crosslinked DHA-MeHA/ODEX hydrogels reached as high as ~<NUM> kPa at about <NUM> of irradiation, which was much higher than both of the DHA-MeHA/ODEX hydrogels without irradiation (dynamic acylhydrazone bond cross-linked HA-dextran hydrogel) and the DHA-MeHA without ODEX components, due to dynamic acylhydrazone crosslinking and chemical crosslinking between methacrylate groups.

Chemical crosslinking can be terminated by simply removing irradiation, and the extent of chemical crosslinking can be tuned by adjusting irradiation exposure time. The G' of such dual-crosslinking hydrogel could be adjust from <NUM>~<NUM> kPa, which was beyond expectation and very surprising for a person skilled in the art.

To quantitatively assess degradation kinetics, HA-dextran hydrogels prepared during Example <NUM>, including the dynamic acylhydrazone bond cross-linked hydrogel and the dual-crosslinked DHA-MeHA/ODEX hydrogel (<NUM> diameter, <NUM> thickness) were incubated in separate wells of a <NUM>-well plate containing <NUM> U/mL exogenous hyaluronidase in <NUM> PBS on an orbital shaker at <NUM>. The solutions were refreshed every <NUM> hours until day <NUM>. Hydrogel samples were removed from the cultures after <NUM>-<NUM> d, washed with distilled water, and lyophilized in a freeze dryer at -<NUM> for <NUM> days and weighed. The extent of degradation was estimated from the weight loss of the polymer based on the following equation: <MAT>.

Where Wo is the original weight of the hydrogel samples, and Wd is the weight of dry sample after being degraded by hydrolysis in PBS.

Degradation kinetics of the dynamic acylhydrazone bond cross-linked hydrogels and dual-crosslinked hydrogels after irradiation prepared during Example <NUM> in the presence of <NUM> U/ml exogenous hyaluronidase for <NUM> d were shown in <FIG>. Dynamic acylhydrazone bond cross-linked hydrogels exhibited rapid HA release, whereas irradiated hydrogels exhibited little HA release, which suggested that secondary polymerization inhibited the degradation of the hydrogels.

Bone marrow stromal cells (BMSCs) were isolated from neonatal SD rats. Cells were cultured in low-glucose Dubecco's Modified Eagle Medium (DMEM, Gibco) supplemented with <NUM>% fetal bovine serum (FBS, Gibco). These cells were located in humidified incubator at <NUM> with a <NUM>% CO<NUM> atmosphere. After <NUM> days, non-adherent cells were removed and fresh media was added. The cells were passaged upon almost confluence. Only three-passage BMSCs were used for hydrogel studies.

The same preparation method as Example <NUM> were performed except that cells were incorporated into the hydrogel in the stage of "Dual-crosslinked DHA-MeHA/ODEX hydrogel formation". Single cell suspensions of BMSCs were mixed with DHA-MeHA and ODEX solutions prepared in Example <NUM>. The final concentration of encapsulated BMSCs in the Dual-crosslinked DHA-MeHA/ODEX hydrogel prepared in Example <NUM> was <NUM> × <NUM><NUM> cells mL-<NUM>. The hydrogels (<NUM>µL each) encapsulated with BMSCs were incubated a <NUM>-well culture plate at <NUM> in <NUM>% CO<NUM> with medium change every two days. Cell viability and proliferation of the encapsulated hASCs were determined by LIVE/DEAD assays according to the manufacturer's protocol (Biovision). For quantitative analysis of LIVE/DEAD result, images were taken randomly from three hydrogel samples at <NUM>, <NUM> and <NUM> d after encapsulation using a Leica inverted microscope. The cell viability was defined as the ratio between the number of live cells and the total cell number in the field.

The cell viability was demonstrated by using LIVE/DEAD assay (n=<NUM>). As shown in <FIG>, the viable stem cells remained above <NUM> % after <NUM> days, which indicated the dual-crosslinked hydrogel are cytocompatible and the photo-irradiation does not harm the cells.

Twelve healthy mature male New Zealand white rabbits weighing around <NUM>-<NUM> were used for cartilage repair experiment. The full thickness cartilage defects with ~<NUM> diameter were created on each knee joint. The empty defects of left cartilage were acted as control group. All surgical procedures were performed under aseptic conditions. The rabbits were anaesthetized by using chloral hydrate (<NUM>/kg body weight). The hind limb was shaved, prepped with betadine and <NUM>% alcohol. A parapatellar, longitudinal incision was made and the patella was luxated medially exposing the femoropatellar groove. Using a dental drill of <NUM> diameter, a circular defect (~<NUM> in diameter and ~<NUM> in depth) was created at the femoro patellar groove by increasing the defect size sequentially to full osteochondral thickness. The drilling was done under continuous irrigation with saline solution for cooling and removing residual tissue while creating the defect. A sterile gauss was used to blot blood at the defect and to obtain homeostasis. Aseptically prepared dynamic acylhydrazone bond cross-linked hydrogel hydrogels prepared during Example <NUM> (without cells) of each rabbit group were loaded into a syringe. The defect was filled with injectable hydrogels (<NUM>µL) using <NUM> needle and then was exposed to <NUM> mW cm-<NUM> <NUM> blue light for <NUM>. The patella was repositioned, followed by suturing the muscle and skin separately. The osteochondral defect was also created in contralateral limb by repeating the same procedure. Bilateral surgery was performed in the articulating knee joints and the approach was identical for all rabbits. Animals were moved to their cages after they recovered from anesthesia. Post and pre-surgical care was taken by intra-muscular administration of penicillin (<NUM>,<NUM> units). At <NUM> weeks, animals were euthanized by carbon dioxide inhalation and the femoral end of knee joints was dissected for further analysis.

As shown in <FIG>, after <NUM> weeks, the hydrogel injected groups exhibited closure of defect with no adverse degeneration of tissue when compared control groups. Moreover, more deposition of newly formed tissue was observed in animals treated with hydrogel group.

As used herein, terms such as "comprise(s)" and the like as used herein are open terms meaning 'including at least' unless otherwise specifically noted. Where a numerical limit or range is stated, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

Claim 1:
A process to prepare a dual-crosslinked hydrogel, comprising the following steps:
<NUM>) functionalizing a glycosaminoglycan such as hyaluronic acid with a methacrylate on hydroxyl group and with a dihydrazide on carboxyl group to obtain a methacrylate and hydrazide group functionalized glycosaminoglycan, which can be either a glycosaminoglycan with both methacrylate and hydrazide group modification, or a glycosaminoglycan with methacrylate modification only and a glycosaminoglycan with hydrazide group modification only, wherein the degree of methacrylation is <NUM>-<NUM>%, preferably <NUM>-<NUM>%, and the degree of hydrazide group modification is <NUM>-<NUM>%, preferably <NUM>-<NUM>%, more preferably <NUM>-<NUM>%; oxidizing a second-polysaccharide such as dextran to obtain a second-polysaccharide with aldehyde groups, the degree of aldehyde modification in the second-polysaccharide is <NUM>-<NUM>%, preferably <NUM>~<NUM>%;
<NUM>) crosslinking the methacrylate and hydrazide group functionalized glycosaminoglycan and the second-polysaccharide-aldehyde in an aqueous solvent, such as phosphate buffer saline or a cell culture medium, to form a dynamic acylhydrazone bond cross-linked hydrogel; and
<NUM>) photopolymerizing the dynamic acylhydrazone bond cross-linked hydrogel by irradiation under the presence of a photoinitiator.