Hydrogel for injection

Abstract

Hydrogel for injection A hydrogel for injection, · Comprising nanocrystalline cellulose oxide and · A gelatin peptide chain, and The nanocrystalline cellulose oxide and the gelatin peptide chain are crosslinked.

Description

Technical Field

[0001] This specification relates to an injectable hydrogel, a method for manufacturing the injectable hydrogel, and the use of the injectable hydrogel for treating diseased joints having a disease such as arthritis.

[0002] Joints are essential for the mobility of humans and animals. They are filled with synovial fluid and include articular cartilage as a complex non-linear viscoelastic anisotropic material that undergoes millions of cycles of joint loading over decades of wear. Diseased joints can cause severe pain and reduced mobility, thus significantly reducing the quality of life of the affected animal or human. For example, diseased joints can have different injuries or diseases such as arthritis (e.g., rheumatoid arthritis, osteoarthritis or other forms of arthritis), but can also have cartilage degeneration, cartilage damage or subchondral defects, resulting in reduced mobility and chronic pain.

[0003] 3D printing has recently been developed to obtain custom-made scaffolds for implantation into damaged joints, for example due to accidents or trauma. Nevertheless, the damaged joint has to be opened to implant the printed scaffold therein, which requires a major surgical procedure, can result in a long recovery time for the patient, and involves the risk that the implant may be rejected by the body or may result in false results such as limited mobility improvement or limited pain relief. In addition, the printed scaffolds are not adapted to cure joint diseases such as osteoarthritis.

[0004] Arthritis such as osteoarthritis (OA) is a musculoskeletal condition and the leading cause of disability worldwide. OA is often referred to as a joint disease associated with cartilage damage and loss, but OA is a much more diverse disease with a complex etiology that affects all tissues within the joint. The main features of OA include progressive loss of the articular cartilage tissue of the joint, inflammation of the synovial tissue, disruption of the characteristics of the synovial fluid, subchondral sclerosis and osteophyte formation at the edges of the joint, which results in chronic pain, joint stiffness and ultimately impaired mobility.

[0005] Hydrogels are known in the art. For example, Chinese Patent No. 110760103 relates to viscoelastic hydrogels and their preparation methods and uses. Balakrishnan et al., Journal of Materials Chemistry B 2013, 1, 5564 discloses that borate-assisted Schiff base formation results in in situ gelling hydrogels. When these hydrogels are disclosed for cartilage regeneration, they are not suitable for reducing the pain of joints with arthritis and increasing mobility.

[0006] The present disclosure relates to an injectable hydrogel comprising the following, · Nanocrystalline cellulose oxide, and · Gelatin peptide chains, and the nanocrystalline cellulose oxide and the gelatin peptide chains are crosslinked.

[0007] Such a hydrogel can be injected into diseased joints having arthritis, such as type IOA, for example, to restore the viscoelastic properties of the joint and reduce pain due to the very high miscibility of the hydrogel in synovial fluid and its favorable viscoelastic properties. Alternatively, the injectable hydrogel of the present invention can be injected into other parts of a mammal (e.g., a damaged eye). The gelatin peptide chains may not be functionalized, i.e., may contain only naturally occurring moieties. The nanocrystalline cellulose oxide may contain an aldehyde moiety or may have a reactive moiety consisting essentially of an aldehyde moiety. The hydrogel can then be prepared in the form of a stable colloid of gel particles in water buffered at physiological pH.

[0008] Advantageously, the nanocrystalline cellulose oxide and the gelatin peptide chains are crosslinked by an imine moiety. The imine or aldimine moiety can preferably result from the direct reaction of the nanocrystalline cellulose oxide with unmodified gelatin peptide chains, for example, by a Schiff base reaction. As a result, it may not be necessary to use a bridge or linker to obtain the crosslinked hydrogel of the present invention.

[0009] Advantageously, the injectable hydrogel contains a buffer solution that maintains the pH of the injectable hydrogel at 7 to 8, preferably 7.2 to 7.6. For example, the buffer solution is Dulbecco's phosphate buffered saline (DPBS), although other buffer solutions close to or within the desired pH range may also be considered. The buffer solution can increase the biocompatibility of the injectable hydrogel when injected into a joint. The buffer solution can also contribute to an efficient cross-linking reaction.

[0010] Advantageously, the concentration of the gelatin peptide chains in the composition before cross-linking is 1.0 to 2.5 weight / volume %, preferably 1.5 to 2.1 weight / volume %, most preferably 1.7 to 1.9 weight / volume %. Alternatively or in combination, the oxidized nanocrystalline cellulose has a pre-cross-linking concentration of 0.1 to 0.5 weight / volume %, preferably 0.2 to 0.4 weight / volume %, most preferably 0.3 weight / volume %. The compositions and hydrogels of the present invention that result in efficient cross-linking can be obtained from any combination of the above concentrations of oxidized nanocrystalline cellulose and gelatin peptide chains.

[0011] Advantageously, the injectable hydrogel contains 0.5 to 2.5 weight / volume % of cross-linked oxidized nanocrystalline cellulose and gelatin (i.e., the gel fraction), preferably 0.8 to 2 w / v %, for example 1.5 or 1.9 w / v %. In other embodiments, the injectable hydrogel can have a concentration of 0.1 to 10.0 (w / v) %, or 0.5 to 5.0 (w / v) %. These concentrations of the hydrogel can enable easy and painless injection into diseased joints while efficiently restoring mobility.

[0012] Advantageously, the injectable hydrogel has an elastic modulus of 10.0 to 220.0 Pa at 0.1 to 10.0 Hz, preferably 50.0 to 200.0 Pa, most preferably up to 80.0 Pa, for example 120.0 Pa. Such an elastic modulus enables optimal treatment of diseased joints.

[0013] Advantageously, the injectable hydrogel has a loss modulus of less than 10.0 Pa, preferably less than 9.0 Pa, between 0.1 and 10.0 Hz. Such a loss modulus allows optimal treatment of pathological joints allowing the restoration of a high level of mobility. For example, the loss modulus may be at least 5.0 Pa, at least 6.0 Pa or at least 7.0 Pa, between 0.1 and 10.0 Hz.

[0014] Advantageously, the injectable hydrogel has a loss modulus to elastic modulus ratio (loss tangent, tan δ) of 10 at 0.1 Hz. 5 Exceeds 10 at 10.0 Hz 3 Such a ratio allows optimal restoration of the viscoelastic properties of the pathological joint.

[0015] Advantageously, the complex viscosity of the injectable hydrogel is less than 1.0 MPa.s, preferably less than 0.9 MPa.s, and even more preferably less than 0.8 MPa.s or less than 0.7 MPa.s.

[0016] Advantageously, the injectable hydrogel has a crosslinking degree of 25-60%, preferably 38-51%, most preferably 42-49%. A higher crosslinking degree may make the hydrogel difficult to inject and may have adverse effects on the diseased joint. A lower crosslinking rate may not heal or alleviate the diseased joint.

[0017] Preferably, the injectable hydrogel is sterile and / or homogenous, and does not contain any aggregates, insoluble particles or solid phases.Furthermore, the injectable hydrogel may be completely homogenous and / or isotropic when mixed with synovial fluid, i.e., may form a stable colloid without solids or supernatants, to limit inflammation and restore mobility to impaired joints.Once crosslinked, the hydrogel may be directly injected.

[0018] Another aspect of the present disclosure is a method for preparing an injectable hydrogel according to any of the preceding claims, the method comprising: - diluting oxidized nanocrystalline cellulose in non-pyrogenic water; - A step of adding a gelatin peptide chain to the diluted nano-crystalline cellulose oxide at room temperature, and - A step of reacting the gelatin peptide chain with the nano-crystalline cellulose oxide.

[0019] The above method may enable the hydrogel of the present invention to be obtained at a limited cost. When the gelatin peptide chain is added at a low temperature, the gelatin peptide chain may result in a non-uniform hydrogel and may have a helical structure that hinders safe injection.

[0020] Advantageously, the reaction step is carried out for 10 to 25 hours and / or at room temperature, for example, 15 to 25 °C. A higher temperature cross-linking reaction may prevent an efficient cross-linking reaction. A low temperature cross-linking reaction may result in a non-uniform hydrogel.

[0021] Advantageously, the reaction step can be carried out at a weakly alkaline pH, for example, greater than 7 and less than or equal to 8, preferably 7.2 to 7.6. A pH buffer, such as a DPBS buffer, can be used, for example, without stirring.

[0022] Advantageously, the gelatin peptide chain is prepared by a dilution step in pyrogenic water at 37 °C, and then the diluted gelatin peptide chain is cooled to room temperature, for example, 20 °C or at least 15 to 25 °C. This enables optimal dilution of the gelatin peptide chain and prepares the gelatin peptide chain for addition to the nano-crystalline cellulose.

[0023] Another aspect of the present disclosure is a medical syringe filled with the above injectable hydrogel. Such a medical syringe is advantageous, for example, for rapid injection into diseased joints having OA. The medical syringe may be pre-filled, that is, filled at the factory before being delivered to the patient and medical staff.

[0024] Preferably, the medical syringe includes a single barrel that defines a reservoir for containing the hydrogel (i.e., after the cross-linking reaction), and a stopper for closing the end of the barrel, and the stopper is movable within the barrel. The medical syringe may preferably include a needle and / or a needle adapter for direct injection of the injectable hydrogel into a diseased joint. The needle may be in the range of 27 gauge to 17 gauge, preferably 27 to 19 gauge, such as 25 to 21 gauge. The hydrogel may be stored in the syringe and / or injected using the syringe in the form of a stable colloid in water buffered to physiological pH, such as pH = 7.2 to 7.4.

[0025] Another aspect of the present disclosure is the use of the above injectable hydrogel for reducing the symptoms of osteoarthritis in mammals such as, for example, horses or humans.

[0026] Another aspect of the present disclosure is a method of treating or curing a diseased joint, particularly a joint having OA, the method comprising the steps of providing the injectable hydrogel disclosed above and injecting such injectable hydrogel into the diseased joint. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Further advantages and preferred embodiments of the present invention will become apparent from the following detailed description and the drawings.

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[0042] The present disclosure includes hydrogels adapted to be injected into joints to increase mobility and reduce, for example, joint pain associated with osteoarthritis. The inventors have studied the changes in the viscoelastic properties of equine synovial fluid (SF) in osteoarthritis and have determined that in joints with osteoarthritis, the rheological properties of the synovial fluid are reduced. In particular, the synovial fluid becomes less viscous and less elastic, and as a result, is less effective in joint lubrication.

[0043] In FIG. 1, the measured values of elastic modulus (G') and viscous modulus (G") as a function of frequency are shown for equine synovial fluid of normal midcarpal joints and diseased midcarpal joints (having type I osteoarthritis). In orthopedically normal SF and pathological SF, it can be observed that the elastic component (G') is dominant over the viscous component (G") for all frequency deformations. However, the absolute values of G' and G" of normal SF are significantly higher compared to diseased SF.

[0044] The hydrogels according to the present disclosure can restore joint lubrication when injected into joints affected by osteoarthritis, and thus improve the mobility of a patient or animal and reduce joint pain.

[0045] The hydrogel may contain, in addition to purified water, at least two components: cellulose and gelatin peptide chains. However, other components such as pH buffers, growth factors, proteins, hyaluronic acid and / or polycaprolactone may be added. Alternatively or in combination, another type of polysaccharide, such as chitosan, can be used to form a hydrogel having gelatin peptide chains.

[0046] Cellulose can be extracted from biomass sources or bacteria. Cellulose is preferably in the form of nanocrystalline cellulose or cellulose nanocrystals, excluding, for example, cellulose nanofibers. Cellulose nanocrystals can have a rod-like shape with distinct faces such as six faces with dimensions of 100 - 900 nm in length, preferably 150 - 850 nm, more preferably 240 - 760 nm, 370 - 620 nm, 410 - 580 nm, and most preferably about 500 nm. The width and height of the crystal can be 5 - 40 nm, preferably 9 - 32 nm, more preferably 10 - 25 nm, and most preferably 15 - 20 nm.

[0047] Nanocrystalline cellulose can be obtained by hydrolysis according to the following Reaction 1 and Figure 2.

Chemical formula

[0048] In Figure 2, cellulose 10 extracted from a biomass source or bacterial source such as cotton, for example, can contain a crystalline domain and an amorphous domain, and the amorphous domain can be cleaved, for example, by acid hydrolysis using sulfuric acid to obtain only the crystalline domain in the form of nanocrystals 11. Depending on the reaction conditions, different shaped nanocrystals can be obtained, while the rod-like shape can provide favorable rheological properties to the composition. In contrast to their low molecular weight homologues, nanocrystalline cellulose can have limited diffusivity due to their high molecular structure, which is advantageous for intra-articular injection.

[0049] Nanocrystalline cellulose may have terminal aldehyde-reactive moieties. In addition, the nanocrystalline cellulose may be functionalized to increase the number of reactive moieties or to add additional reactive moieties. As a result, the number or type of additional reactive moieties may be different from the reactive moieties of natural and / or nanocrystalline cellulose. Preferably, the reactive moiety comprises a C=O double bond and is preferably an aldehyde moiety. These aldehyde moieties can be obtained by oxidation of nanocrystalline cellulose with a strong oxidizing agent such as periodate, as shown in Reaction 2 below.

Chemical formula

[0050] According to Reaction 2, the periodate ion can be linked to two hydroxyl moieties of the sugar ring of cellulose by elimination of a water molecule. Electron rearrangement results in ring opening of the sugar ring, thus providing a dialdehyde chain instead of the sugar ring. Thus, the reactive moiety is preferably an aldehyde moiety, e.g., mainly an aldehyde moiety or an aldehyde moiety only.

[0051] Thus, oxidation at positions C2 and C3 can result in cleavage of the sugar ring, which changes the structure of the polysaccharide chain. An increase in the backbone flexibility of the polymer can be obtained. Stereostabilized nanocrystalline cellulose can be obtained by an oxidation process that introduces flexible units into the polysaccharide chain. The flexible units may enable or contribute to the colloidal stability of the hydrogel. Here, the dialdehyde units formed by the oxidation reaction can be in equilibrium with other units, as seen in Figure 3.

[0052] Figure 3 represents different cycles or units of cellulose that can be formed after the above oxidation reaction. Figure 3(a) shows the free aldehyde moiety, Figure 3(b) shows the intramolecular hemiacetal moiety, Figure 3(c) shows the hemiaminal moiety, Figure 3(d) shows the hydrated aldehyde, i.e., the hydroxyl moiety generated by the hydration of the aldehyde moiety, and Figure 3(e) shows the intermolecular hemiacetal. The intermolecular hemiacetal in Figure 3(e) can contribute to the cross-linking between the chains of nanocrystalline cellulose, but these hemiacetal cross-links are rare and mostly occur between the chains of the same cellulose nanocrystal.

[0053] The aldehyde content can be evaluated by UV / vis spectrophotometry (BCA assay) using D-(+)-glucose as a standard. For example, the aldehyde content is 800 - 3500 μmol / g, preferably 1000 - 2800 μmol / g, more preferably 1400 - 2200 μmol / g, and most preferably about 1900 μmol / g of the oxidized nanocrystalline cellulose.

[0054] The gelatin can be any gelatin compatible with the human or animal species to be treated. In particular, type B gelatin can be used. The gelatin is preferably not functionalized and may contain only naturally occurring reactive moieties as seen in Figure 4.

[0055] The gelatin may preferably contain gelatin peptide chains obtained from bovine gelatin because the isoelectric point of bovine gelatin is close to that of the human body. However, other types of gelatin may be used, especially for non-human applications. Figure 4 shows the (partial) structure of gelatin, showing, for example, arginine, glycine, and proline. However, as known to those skilled in the art, other natural amino acids may be present in the gelatin.

[0056] The hydrogel of the present invention can be crosslinked by the reaction between the oxidized nanocrystalline cellulose and the gelatin peptide chain, in particular, by the reaction between the nucleophilic moiety of gelatin and the aldehyde moiety of the oxidized nanocrystalline cellulose. For example, crosslinking can be achieved via a Schiff base reaction according to Reaction 3.

Chemical formula

[0057] According to Reaction 3, the ε - amine moieties of the lysine and hydroxylysine amino acids of gelatin can react with the available aldehyde moieties of the oxidized nanocrystalline cellulose, thus resulting in imine moieties that link the peptide chains and sugar chains of gelatin and nanocrystalline cellulose respectively. This reaction is preferably carried out at room temperature. In fact, room temperature enables the efficient production of a pure hydrogel ready for injection. In contrast, the inventors have found that the use of higher temperatures may prevent an efficient crosslinking reaction.

[0058] For example, the molar ratio of gelatin to oxidized nanocrystalline cellulose may be 1.8 - 0.2, preferably 1.5 - 0.5, more preferably 0.8 - 1.2, and most preferably 1. The degree of crosslinking can be defined as the ratio of the amount of substance of the imine moiety in the hydrogel to the amount of substance of the aldehyde moiety on the oxidized cellulose before forming the hydrogel. The degree of crosslinking may be at most 70% or at most 60%, preferably 38 - 55%, more preferably 45 - 53%, for example about 49%. Such a degree of crosslinking enables good injectability.

[0059] This hydrogel may be rheopectic. For example, the viscosity of the hydrogel can be 100 - 500 Pa·s at a shear rate of 0.1 s -1 and after 100 s.

[0060] Rheological analysis The investigation of rheological behavior was carried out using an Anton Paar Modular Compact Rheometer MCR 302e. A plate-plate geometry (PP25) with a gap of 0.5 mm was used. The properties of the sample were examined at the temperature of the horse, i.e., 37.5 °C, with an accuracy of 0.1 °C provided by the Peltier plate temperature control system of the instrument.

[0061] Vibration test and viscoelasticity The viscoelastic properties of the sample were determined by dynamic experiments. The storage modulus (or elastic modulus) (G’), loss modulus (or viscous modulus) (G”), loss tangent (tanδ), and complex viscosity (η*) were monitored as functions of frequency.

[0062] The loss tangent (tanδ) is defined according to Equation 1.

Equation

[0063] The complex viscosity η* is a measure of the total resistance to flow as a function of the angular frequency (ω) and is given by the quotient of the maximum stress amplitude and the maximum strain rate amplitude.

[0064] The dynamic strain sweep test was also carried out as follows: At a fixed frequency, a strain sweep from 0.1 to 100% was applied to determine the linear viscoelastic range. Dynamic frequency sweep test: The strain constant was maintained at 10%, and a frequency sweep from 0.1 to 4 Hz was applied. The frequency range studied included the physiological frequencies of knee movement (0.5 Hz for slower knee movements, up to 3 Hz).

[0065] Rotation test and viscosity The synovial fluid sample was subjected to rotation and viscosity tests at a constant shear rate of 0.01 s -1 The test was immediately after a pre-shear of 60 seconds at 100 s -1 and t = 0 was immediately after the end of the pre-shear and the start of the application of 0.01 s -1 for 600 seconds. The shear stress τ follows Equation 2. Where F is the shear force (unit N) and A is the shear area A (unit m 2) is as follows.

Number

[0066] The viscoelastic properties (loss G” and elastic G modulus) of a 2.0% w / v injectable hydrogel as a function of frequency are shown in Fig. 5, and the complex viscosity and tanδ dependence on frequency are reported in Fig. 6. The rheological behavior of the hydrogel of the present invention is typical of a weak gel. Over the entire frequency range, the elastic modulus (G’) is always about one order of magnitude higher than the viscous modulus (G”), and both elastic moduli are independent of frequency.

[0067] Furthermore, the elastic modulus G’ always exceeds 10 Pa at 0.1 to 10.0 Hz, preferably exceeds 50 Pa, and more preferably exceeds 80 Pa. The loss elastic modulus G’’ is always less than 10 Pa, preferably less than 9 Pa, in the same frequency range.

[0068] Regarding Fig. 6, the loss coefficient (tanδ) is 10 at 0.1 Hz 5 or more, 10 at 1 Hz 4 or more, and 10 at 10 Hz 3 or more. Therefore, the loss coefficient may be 10 3 to 10 5 in the frequency range of 0.1 to 10 Hz. The complex viscosity may be 10 -2 to 1 MPa·s, preferably 0.2 to 0.8 MPa·s, and more preferably about 0.1 MPa·s.

[0069] In vitro results To demonstrate the hardening properties of the hydrogel of the present invention on arthritic joints, the hydrogel of the present invention at a concentration of 2% (w / v) in PBBS buffer was studied in a mixture with equine synovial fluid (10% by volume of the hydrogel in diseased synovial fluid, i.e., 1 mL of hydrogel in 9 mL of synovial fluid) to mimic what occurs in vivo after injection. Figure 7 shows the elastic (G') and viscous (G") moduli as a function of frequency from diseased synovial fluid (OA SF) and diseased synovial fluid mixed with 10% of the above hydrogel (v / v), i.e., 1 mL of hydrogel in 9 mL of diseased synovial fluid. As can be seen in Figure 7, the viscoelastic modulus values of diseased synovial fluid (OA SF) increase for all deformation frequencies when mixed with the hydrogel of the present invention.

[0070] Figure 8 shows the elastic (G') and viscous (G") moduli as a function of frequency of diseased synovial fluid and normal synovial fluid (normal SF) (both extracted from equine metacarpophalangeal joints) mixed with 10% hydrogel 2.0% (w / v). This experiment makes it possible to mimic diseased joints healed by the hydrogel of the present invention with respect to normal joints. The values of the elastic (G') and viscous (G") moduli of diseased synovial fluid mixed with the hydrogel of the present invention are close to or similar to the values obtained with synovial fluid from normal and healthy joints. These results allow the conclusion that the hydrogel of the present invention can be successfully injected into diseased joints with osteoarthritis to relieve pain and at least partially restore mobility.

[0071] Figure 9 shows the shear stress τ as a function of time for diseased synovial fluid and diseased synovial fluid mixed with 10% (v / v) of the hydrogel 2.0% (w / v) of the present invention. Figure 9 shows that both samples exhibit rheological behavior defined as very close shear stress values and shear stress increasing over time at a constant shear rate.

[0072] Figure 10 shows the results of the same experiment as in Figure 7, but using synovial fluid from a different joint: the equine calcaneocuboid joint. The elastic modulus of both pathological synovial fluids is low, but the loss elastic modulus G” rapidly decreases above 1 Hz (i.e., during fast walking and running). In contrast, when mixed with 10% (v / v) of the hydrogel of the present invention at 2% (w / v), both elastic moduli increase. At low frequencies (i.e., walking), the mixed synovial fluid maintains its viscous properties, but at higher frequencies (running), the mixed synovial fluid tends to maintain its elastic properties and potentially protects the articular cartilage from unrelieved forces.

[0073] Next, a detailed example of a procedure that can be used to obtain the hydrogel of the present invention is shown below. These procedures do not limit the general disclosure above and are provided to enable sufficient disclosure.

[0074] Preparation of oxidized nanocrystalline cellulose Add 300 mL of deionized water to a 1 L two-necked flask and acidify to pH 3.0 with 5.5 mL of acetic acid (36%). Suspend 6.0 g of cellulose nanocrystals under mechanical stirring. Add 92.5 mL of a 0.7 M NaIO4 solution dropwise to the mixture. Protect the flask from light and react the mixture at 45 °C for 4 hours.

[0075] Add ethylene glycol (5.0 mL) to stop the reaction, adjust the pH to 6.0 with Na2CO3 (1 M, 17.3 mL), and stir for an additional 1 hour. Dialyze the final mixture against water (28 L) at room temperature for 5 days (SpecPor5, 12 - 14 kDa), changing the water three times until the total dissolved solids content (TDS) reaches 0 ppm.

[0076] Preparation example of 2.0% (w / v) hydrogel Dilute a 3.2 mL suspension of dialdehyde nanocrystalline cellulose (aldehyde content: 1891 μmol / g) with pyrogen-free water to a total volume of 10 mL. Dilute 170 mg of gelatin type B (primary amine content: 435 μmol / g) in pyrogen-free water at a temperature of 37 °C and then cool to 20 °C. Add the diluted gelatin to the dialdehyde nanocrystalline cellulose at a temperature of 20 °C together with 96 mg of Dulbecco's phosphate buffered saline (DPBS) to buffer the solution to pH 7.2 - 7.4. No stirring was performed during crosslinking.

[0077] The crosslinking reaction is complete after 20 hours at room temperature, thereby obtaining a viscoelastic hydrogel. The hydrogel is maintained at a temperature of 15 - 25 °C during storage and then irradiated with gamma rays, such as from a gamma ray source of 30 kGy, i.e., at least 25 kGy of C 60 It may be sterilized by gamma irradiation, such as from a gamma ray source. The effectiveness of the crosslinking reaction (Schiff base reaction) is evaluated by the TNBS assay to determine the concentration of primary amines. As an alternative method, acid-base titration with hydroxylamine was also performed. Based on this data, the inventors can conclude that all aldehydes reacted quantitatively with amines, resulting in a degree of crosslinking of 49.9% of the gelatin.

[0078] Determination of aldehyde groups in BCA method, oxidized nanocrystalline cellulose The nanocrystalline cellulose sample is diluted to a concentration of 0.01% with 0.01% BCA buffer (1.0 M KCl; 0.01 M Na2CO3; 0.09 M NaHCO3). For the calibration curve, 0.09008 g of D-(+)-glucose is dissolved in a 500 mL volumetric flask with BCA buffer to obtain a 1000 μM D-(+)-glucose stock solution. A standard series is prepared by diluting the stock solution to the following concentrations: 0, 50, 100, 250, 500 μM. Standards such as the diluted samples are processed. 100 μL of the diluted sample / standard is transferred to a glass vial with a cap, and 900 μL of BCA buffer is added. Next, 1000 μL of freshly prepared BCA reagent solution (0.2561 M Na2CO3; 0.1440 M NaHCO3; 0.0025 M BCA; 0.0039 M CuSO4·5H2O; 0.0060 M) is added. Then, the glass vial is heated in a water bath at 75 °C for 30 minutes. The absorbance is measured with a double-beam spectrophotometer at a wavelength of 560 nm against deashed H2O.

[0079] The aldehyde content is preferably 1500 - 2500 μmol / g of nanocrystalline cellulose, preferably 1700 - 2300 μmol / g, more preferably 1800 - 1900 μmol / g.

[0080] Determination of free primary amino groups in crosslinked hydrogel samples by the TNBS method 10 μL of the sample is transferred to an amber glass vial with a screw cap, and 990 μL of TNBS buffer (0.1 M NaHCO3; pH 8.5) is added. The gelatin concentration of the diluted sample was approximately 0.01% (w / v). To draw a calibration curve, a stock solution is prepared by weighing 0.0563 g of L-glycine with an analytical balance and dissolving it in a 500 mL volumetric flask with TNBS buffer to a final concentration of 1500 μM L-glycine. The stock solution is then diluted with TNBS buffer to prepare a standard series with the following concentrations: 10, 25, 50, 75, 100, 150 μM. For each standard, 1000 μL is transferred to an amber glass vial with a screw cap and processed in the same manner as the sample.

[0081] The amine content is preferably 220 to 840 μmol / g of the gelatin peptide chain, preferably 350 to 520 μmol / g, and most preferably 390 to 450 μmol / g.

[0082] A 0.05% TNBS solution was freshly prepared by diluting 5% TNBS with TNBS buffer. 500 μL of the freshly prepared 0.05% TNBS solution was added to each vial. The solution was incubated in an oven at 37 °C for 2 hours. After incubation, 250 μL of 1 M HCl and 500 μL of deionized H2O were added to each vial to stop the reaction. Then, all the solutions were hydrolyzed in an autoclave at 120 °C and about 15 psi (103421 Pa) for 1 hour. After hydrolysis, about 2 mL of the solution was transferred to a UV cuvette with a path length of 1 cm and measured with a double-beam spectrophotometer at a wavelength of 335 nm against a blank prepared in the same manner as the sample.

[0083] Crosslinking reaction To investigate the efficiency of the crosslinking reaction, a first composition (Example of FIG. 11) containing the above gelatin peptide chain and oxidized nanocrystalline cellulose was prepared, and a second composition (Comparative Example 1 of FIG. 11) containing the above gelatin peptide chain and non-oxidized nanocrystalline cellulose, i.e., only natural aldehyde moieties, was prepared. Both compositions were incubated at 20 °C without stirring, and the elastic modulus of both compositions was investigated over time (i.e., up to 25 hours).

[0084] As seen in FIG. 11, the elastic modulus G' for Comparative Example 1 increased slightly over time, indicating physical gel formation. In contrast, the elastic modulus G' for the Example (i.e., the hydrogel of the present invention at 2% w / v) increased strongly over time, indicating physical gel formation and efficient crosslinking. Thus, by using oxidized nanocrystalline cellulose, it is possible to obtain appropriate rheological and mechanical properties of the hydrogel of the present invention by efficient crosslinking.

[0085] Next, a second experiment was conducted to determine the effect of temperature on the cross-linking reaction. The increase in the elastic modulus G' over time was investigated for the above-described first composition and a third composition identical to the first composition. The first composition was incubated at 20 °C without stirring (Example in Fig. 12), while the third composition was incubated at 40 °C (Comparative Example 2 in Fig. 12).

[0086] As can be seen in Fig. 12, the elastic modulus G' of Comparative Example 2 did not increase over time, indicating that there was no cross-linking or that cross-linking was very limited. In contrast, the elastic modulus G' for the Example (i.e., the hydrogel of the present invention at 2% w / v) increased strongly over time, indicating efficient cross-linking. Therefore, the optimal temperature for efficient cross-linking is 15 - 25 °C, preferably about 20 °C.

[0087] Next, considering the elastic modulus G' obtained at 20 °C with the resulting hydrogel (2% w / v), the effect of pH on the cross-linking reaction was investigated. The imine moiety is formed when the primary amine moiety reacts with an aldehyde or ketone moiety under appropriate conditions. Imine formation usually requires an acid catalyst. The acid catalyst may enable the removal of water. On the other hand, this mechanism involves a deprotonated amine for nucleophilic attack on the carbonyl functional group.

[0088] Fig. 13 shows the elastic modulus (G') of a 2% w / v hydrogel obtained from the cross-linking reaction at different pH buffers and different reaction times (0 - 25 hours). The elastic modulus was measured as disclosed above. After only 10 hours of reaction, higher elastic moduli are obtained at pH 6.0 and 7.0. The cross-linking reaction in a phosphate buffer at pH = 6.0 results in a hydrogel having a higher elastic modulus compared to pH = 7.0 at a given reaction time, supporting the fact that mild acidic conditions are favorable for imine formation.

[0089] For the hydrogel obtained at pH = 8, a strong increase is shown initially (<5 h), but the storage modulus G’ then levels off, presumably due to the alkaline degradation of the hydrogel. For the hydrogel obtained at pH = 5, the crosslinking reaction has a limited rate, and the storage modulus increases with reaction time but cannot match that obtained with the hydrogels obtained at pH = 6 or 7 for a given reaction time exceeding 5 h. Considering the desired storage modulus G’, the optimal pH for the crosslinking reaction can be between 6.0 and 7.5. A crosslinking pH of 7.4 enables the formation of gels under physiological conditions with a high crosslinking rate, thus avoiding an additional step of changing the hydrogel pH.

[0090] In vitro toxicity As shown in Table 1, in vitro cytotoxicity was performed on 1.0 and 2.0 (w / v)% dialdehyde nanocrystalline cellulose, nanocrystalline cellulose, and the hydrogels of the present invention according to the method of ISO 10993-5:2009. This demonstrates that the hydrogels of the present invention, in contrast to dialdehyde nanocrystalline cellulose, have no cytotoxic effect and can be safely injected.

Table 1

[0091] Miscibility Body joints are filled with synovial fluid, and miscibility is an important criterion for evaluating the ability of hydrogels to treat joints with OA. Synovial fluid is mainly composed of water, hyaluronan, lubricin, proteinase, collagenase, and prostaglandin and has a pH of about 7.5.

[0092] Injection of immiscible hydrogels into the joints of the disorder to be treated may not provide benefits regarding pain and mobility, or may provide limited benefits, but may induce a strong inflammatory response, and thus, decompose the treated joints. As a result, the miscibility of the hydrogels of the present invention was evaluated under physiological conditions (i.e., PBS buffer, pH = 7.4 at a temperature of 37 °C) and compared with the miscibility of the comparative hydrogels described below. The results of this evaluation are shown in Table 2.

[0093]

Table 2

[0094] The hydrogels of the present invention are completely miscible under physiological conditions and form stable colloids at a concentration of 1% to 4% w / v. The same miscibility results were observed in equine synovial fluid (see the above rheology and viscoelasticity experiments). Without being bound by any theory, it is considered that the unique chemical structure of the hydrogel enables the formation of stable colloids in PBS buffer and synovial fluid, and thus, a single phase having favorable viscoelastic and rheological properties can be obtained.

[0095] Comparative Example Comparative Example 3 was obtained by replacing gelatin with collagen in the above-described hydrogel preparation method. The conditions for the cross-linking reaction between collagen and oxidized nanocrystalline cellulose were a temperature of 20 °C without stirring for 10 hours. Under the above conditions, the cross-linking reaction did not occur and no hydrogel was obtained.

[0096] Comparative Example 4 relates to a hydrogel prepared according to Example 3 of Chinese Patent No. 110760103. The hydrogel is prepared by mixing nanocrystalline cellulose oxide at a concentration of 12 mg / mL and pH = 8 with a prepolymerization solution of collagen at the same weight, a concentration of 12 mg / mL and pH = 5, in a container. After slowly mixing at room temperature for 30 minutes, a thick hydrogel is obtained. However, the hydrogel of Comparative Example 4 is immiscible in PBS buffer at pH = 7.4 or synovial fluid, and two different phases are obtained. As a result, rheological measurements as described above cannot be performed.

[0097] In vivo evaluation of the hydrogel of Comparative Example 4 was performed. When a 2% w / v hydrogel according to Comparative Example 4 was subcutaneously injected into mice, no inflammatory reaction was observed. Then, the same 2% hydrogel was injected into three fetlock joints of horses with OA, and a strong inflammatory response was observed. Therefore, it is clear that the hydrogel of Comparative Example 4 cannot reduce the symptoms of joints with OA.

[0098] Comparative Example 5 relates to preparing a hydrogel according to the method of the present invention and replacing nanocrystalline cellulose oxide with carboxymethyl cellulose oxide. Under the above cross-linking reaction conditions, a hydrogel is obtained. However, this hydrogel is immiscible in PBS buffer at pH = 7.4 and immiscible in synovial fluid. As a result, the hydrogel of Comparative Example 5 cannot relieve the symptoms of joint disorders.

[0099] Comparative Example 6 relates to performing a cross-linking reaction between nanocrystalline cellulose oxide and gelatin according to this method. However, the cross-linking conditions were changed to 37 °C in PBS buffer at pH = 7.4. Under these conditions, cross-linking was not obtained and no hydrogel was formed. Without being bound by any theory, it is considered that the reaction rate of β-elimination is greater than the reaction rate of Schiff base formation, thus hindering an efficient cross-linking reaction.

[0100] In vitro stability The in vitro stability of the 2% w / v hydrogel was investigated over 6 months under physiological conditions. Six samples of 2% w / v hydrogel were prepared and maintained at 37 °C and pH = 7.4 while gently shaken. To determine the weight of the hydrogel for each sample, the sample was lyophilized to remove the liquid phase and the weight (wt) of the gel fraction was obtained from a precision scale. After 2 months, the remaining weight was still higher than 90% of the original weight (wo). After 6 months, approximately 20% of the original weight (wo) still remained.

[0101] Therefore, this preliminary stability experiment demonstrated good stability of the hydrogel under physiological conditions and thus supported long-term reduction of OA symptoms in vivo. Pharmacology

[0102] Small equine joints can be treated with 2 mL of 2% w / v hydrogel, while larger equine joints can be treated with 4 mL of 2% w / v hydrogel. For human joints, 1 mL of 2% w / v hydrogel can be used.

[0103] Although the invention has been described and illustrated in detail, this is by way of example and illustration only and should not be construed as limiting, and it is clearly understood that the scope of the invention is limited only by the terms of the appended claims.

Claims

1. A hydrogel for injection, • Oxidized nanocrystalline cellulose and • Contains gelatin peptide chains, A hydrogel for injection, wherein the oxidized nanocrystalline cellulose and the gelatin peptide chain are crosslinked.

2. The injectable hydrogel according to claim 1, wherein the oxidized nanocrystalline cellulose and the gelatin peptide chain are crosslinked by an imine moiety.

3. The injectable hydrogel according to any one of claims 1 to 2, wherein the gelatin peptide chain has a concentration of 1.0 to 2.5% by weight / volume before crosslinking, and / or the oxidized nanocrystalline cellulose has a concentration of 0.1 to 0.5% by weight / volume before crosslinking.

4. The injectable hydrogel according to claim 1, wherein the injectable hydrogel contains 0.5 to 2.5% by weight / volume of cross-linked oxidized nanocrystalline cellulose and gelatin peptide chains.

5. The injectable hydrogel according to claim 1, wherein the injectable hydrogel has an elastic modulus of 10 to 220 Pa at 0.1 to 10.0 Hz.

6. The injectable hydrogel according to claim 1, wherein the injectable hydrogel has a loss modulus of elasticity of less than 10 Pa at 0.1 to 10.0 Hz.

7. The aforementioned hydrogel for injection is 10 at 0.1 Hz. ５ The injectable hydrogel according to claim 1, having a ratio (tanδ) of loss modulus to modulus of elasticity exceeding .

8. The injectable hydrogel according to claim 1, wherein the complex viscosity of the injectable hydrogel is less than 1.0 MPa.s at 0.1 to 10.0 Hz.

9. The injectable hydrogel according to claim 1, wherein the injectable hydrogel has a degree of crosslinking of 25 to 60%, preferably 38 to 49%.

10. A method for preparing an injectable hydrogel according to Claim 1, wherein the method is: - A step of diluting oxidized nanocrystalline cellulose in non-exothermic water, - A step of adding gelatin peptide chains to the diluted oxidized nanocrystalline cellulose at room temperature, A method comprising the step of reacting the gelatin peptide chain with the oxidized nanocrystalline cellulose.

11. The method for preparing an injectable hydrogel according to claim 9, wherein the reaction step is carried out for 10 to 25 hours, preferably at a temperature of 15 to 25°C.

12. A method for preparing an injectable hydrogel according to claim 9, wherein the reaction step is carried out at a pH greater than 7 and less than or equal to 8.

13. A method for preparing an injectable hydrogel according to claim 9, further comprising the steps of diluting the gelatin peptide chain in water at 37°C and cooling the diluted gelatin peptide chain at room temperature.

14. Use of the injectable hydrogel according to claim 1 for treating or alleviating the symptoms of osteoarthritis.

15. A syringe containing the hydrogel described in Claim 1.

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