Hydrogel for osteoarthritis repair and preparation method and application thereof
By crosslinking PDRN with PGA to form a porous hydrogel, the problem of rapid degradation of PDRN in vivo is solved, achieving long-lasting sustained release and high mechanical strength, which significantly improves the treatment effect of osteoarthritis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANDONG ACADEMY OF PHARMACEUTICAL SCIENCES
- Filing Date
- 2025-12-09
- Publication Date
- 2026-06-19
AI Technical Summary
In existing technologies, polydeoxyribonucleic acid (PDRN) has an extremely short half-life in vivo and is easily degraded by nucleases, resulting in low bioavailability and difficulty in maintaining long-term effective therapeutic concentrations. Furthermore, existing treatment strategies are unable to achieve fundamental repair and regeneration of cartilage tissue.
By crosslinking polydeoxyribonucleotides (PDRN) with polyglutamic acid (PGA), a hydrogel with a porous structure is formed. The mechanical strength of the hydrogel is enhanced by the amidation reaction between the PGA side chains and the amino groups of PDRN, and the slow and controlled release of PDRN is achieved, thus prolonging its action time in the joint cavity.
It achieves long-term sustained release of PDRN, enhances the mechanical strength of the hydrogel, and can continuously exert anti-inflammatory effects within the joint cavity, significantly improving the treatment effect of osteoarthritis and meeting the mechanical requirements of the joint weight-bearing area.
Smart Images
Figure CN121338039B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a hydrogel for osteoarthritis repair, its preparation method, and its application. Background Technology
[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] Osteoarthritis, a prevalent degenerative joint disease, has always presented a significant clinical challenge in its treatment. Current treatment strategies, ranging from oral nonsteroidal anti-inflammatory drugs (NSAIDs) to intra-articular injections of hyaluronic acid or corticosteroids, largely focus on short-term symptom relief and are insufficient for achieving fundamental repair and regeneration of cartilage tissue. Therefore, the development of novel therapeutic materials that can actively promote cartilage repair and provide long-lasting therapeutic effects has become an urgent need in this field.
[0004] Polydeoxyribonucleotides (PDRNs), as bioactive substances with clear anti-inflammatory and tissue regeneration functions, have shown great potential in cartilage repair. However, the application of PDRNs in vivo is severely limited by their inherent defects: their half-life in the physiological environment is extremely short, about 3 hours, and they are easily degraded by nucleases, resulting in low bioavailability and difficulty in maintaining long-term effective therapeutic concentrations.
[0005] Therefore, how to provide a regenerative material that combines high mechanical strength, long-lasting sustained release, and osteoarthritis repair functions is an urgent problem to be solved. Summary of the Invention
[0006] In view of this, the present invention provides a hydrogel for osteoarthritis repair, its preparation method and application. The present invention increases the mechanical strength of the hydrogel by crosslinking polydeoxyribonucleotide (PDRN) with polyglutamic acid (PGA). At the same time, PDRN can be slowly released in the hydrogel, which has anti-inflammatory effect and has a significant therapeutic effect on osteoarthritis.
[0007] In a first aspect, the present invention provides a hydrogel for the repair of osteoarthritis, the hydrogel being made of polydeoxyribonucleotides and polyglutamic acid, the hydrogel having a porous structure with an average pore size of 50-200 μm.
[0008] In one or more embodiments, the hydrogel has a compressive modulus of 5-90 kPa.
[0009] A second aspect of the present invention provides a method for preparing the hydrogel for osteoarthritis repair described in the first aspect, comprising the following steps:
[0010] (1) Prepare PDRN solution and PGA solution respectively;
[0011] (2) Mix the PDRN solution with the PGA solution to obtain a pre-crosslinked solution.
[0012] (3) Add cross-linking agent solution to pre-cross-linking solution, stir and mix evenly, and let stand to react.
[0013] In one or more embodiments, in step (1), the solvent in the PDRN solution is selected from one of PBS buffer solution, physiological saline, and deionized water, and the mass concentration of the PDRN solution is 0.01%-4%;
[0014] In step (1), the solvent in the PGA solution is selected from one of PBS buffer solution, physiological saline, and deionized water, and the mass concentration of the PGA solution is 5%-50%.
[0015] In one or more embodiments, in step (1), the crosslinking agent is selected from 1 Ethyl Carbodiimide hydrochloride, N One or more of the following: hydroxysuccinimide, 1,4-butanediol diglycidyl ether, glutaraldehyde, carbodiimide, genipin, cystamine, hexanoisocyanate, diphenylphosphine azide, and polyethylene glycol diacrylate.
[0016] In one or more embodiments, in step (1), the molecular weight of the PGA is 50-300 kDa.
[0017] In one or more embodiments, in step (2), the volume ratio of the PDRN solution to the PGA solution is 1:1-3, preferably 1:1.
[0018] In one or more embodiments, in step (3), the amount of crosslinking agent solution added is 5-50 μL / ml of the pre-crosslinking solution volume.
[0019] In one or more embodiments, in step (3), the reaction temperature is 20-40℃ and the reaction time is 4-12h; preferably, the reaction temperature is 30-40℃ and the reaction time is 6-8h; more preferably, the reaction temperature is 35℃ and the reaction time is 6h.
[0020] Thirdly, the present invention provides the application of the hydrogel described in the first aspect or the hydrogel prepared by the preparation method described in the second aspect in the preparation of osteoarthritis repair drugs.
[0021] Compared with the prior art, the present invention has achieved the following beneficial effects:
[0022] (1) This invention constructs a hydrogel with high mechanical strength, long-lasting sustained release, and osteoarthritis repair function through chemical cross-linking of PDRN and PGA. The PGA side chain is rich in carboxyl groups, which can undergo amidation reaction with the amino groups on PDRN to achieve stable cross-linking through amide bonds, thereby enhancing the mechanical strength of the hydrogel. Because PDRN and PGA form stable chemical bonds, the release of PDRN must depend on the swelling and degradation process of the hydrogel network, which causes the release behavior of PDRN to change from rapid diffusion to slow and controllable release, extending its effective action time in the joint cavity from several hours to several days or even several weeks, thus enabling it to exert a sustained anti-inflammatory effect. This effectively solves the problems of PDRN being easily and rapidly degraded in vivo, having low bioavailability, and being difficult to maintain a long-term effective therapeutic concentration, providing a reliable guarantee for the long-term effective treatment of osteoarthritis.
[0023] (2) Through the chemical cross-linking of PDRN and PGA, the mechanical strength of the hydrogel is significantly enhanced, enabling it to meet the mechanical requirements of the joint load-bearing area. Furthermore, experiments have demonstrated that the hydrogel prepared by this invention has anti-inflammatory effects and a significant therapeutic effect on osteoarthritis. Attached Figure Description
[0024] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.
[0025] Figure 1 This is a SEM image of the hydrogel prepared in Example 1 of the present invention;
[0026] Figure 2 The infrared spectrum of the hydrogel prepared in Example 1 of this invention;
[0027] Figure 3 This is a diagram showing the in vitro release of PDRN from the hydrogel prepared in Example 1 of this invention;
[0028] Figure 4 The graph shows the cell proliferation experiment and anti-inflammatory activity data of the hydrogel prepared in Example 1 of the present invention, where a is the cell proliferation experiment and b is the anti-inflammatory activity.
[0029] Figure 5 The graph shows the concentration data of TNF-α, IL-1β and IL-6 in the synovial fluid of animal joints of the hydrogel prepared in Example 1 of the present invention, where a is TNF-α, b is IL-1β and c is IL-6. Detailed Implementation
[0030] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0031] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not have any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used. Unless otherwise stated, the mass concentration of the solution in the present invention refers to a mass-volume percentage, expressed as %.
[0032] Example 1
[0033] This embodiment provides a hydrogel for osteoarthritis repair and its preparation method.
[0034] (1) Solution preparation: Weigh 0.10 g of PDRN and dissolve it in 5 ml of PBS buffer to obtain a PDRN solution with a mass concentration of 2% (i.e., 0.02 g / ml PDRN solution). Weigh 1.5 g of PGA (106 kDa) and dissolve it in 5 ml of PBS buffer to obtain a PGA solution with a mass concentration of 30% (i.e., 0.3 g / ml PGA solution).
[0035] (2) Take 5 ml of PDRN solution and mix it with 5 ml of PGA solution. Stir magnetically for 15 minutes to ensure that the mixture is homogeneous.
[0036] (3) Take 0.15 ml of 1,4-butanediol diglycidyl ether and slowly add it dropwise to the above mixture to make the amount of crosslinking agent added 15 μL / ml. Continue stirring and maintain the temperature at 35°C, and let it stand for 6 hours.
[0037] Example 2
[0038] The difference between this embodiment and Embodiment 1 is that the molecular weight of PGA is 72kDa, while all other conditions are the same.
[0039] Example 3
[0040] The difference between this embodiment and Embodiment 1 is that the molecular weight of PGA is 130 kDa, while all other conditions are the same.
[0041] Example 4
[0042] The difference between this embodiment and Embodiment 1 is that the molecular weight of PGA is 200 kDa, while all other conditions are the same.
[0043] Example 5
[0044] The difference between this embodiment and Example 1 is that 1.0 g of PGA was weighed and dissolved in 5 ml of PBS buffer, and the mass concentration of the PGA solution was 20% (i.e., 0.2 g / ml PGA solution), while all other conditions were the same.
[0045] Example 6
[0046] The difference between this embodiment and Example 1 is that 2.0 g of PGA was weighed and dissolved in 5 ml of PBS buffer, and the mass concentration of the PGA solution was 40% (i.e., 0.4 g / ml PGA solution), while all other conditions were the same.
[0047] Example 7
[0048] The difference between this embodiment and Example 1 is that 0.2 g of PDRN was weighed and dissolved in 5 ml of PBS buffer, and the mass concentration of the PDRN solution was 4% (i.e., 0.04 g / ml PDRN solution), while all other conditions were the same.
[0049] Comparative Example 1
[0050] The difference between this comparative example and Example 1 is that PDRN is not added in this comparative example, but all other conditions are the same.
[0051] Comparative Example 2
[0052] The difference between this comparative example and Example 1 is that no PGA was added in this comparative example, while all other conditions were the same.
[0053] Performance testing
[0054] Comparative Example 2 could not form a stable hydrogel, therefore performance data could not be measured.
[0055] 1. The morphology and structure of the hydrogel prepared in Example 1 were characterized using scanning electron microscopy (SEM). For example... Figure 1 As shown, the PGA-PDRN hydrogel has a porous structure with an average pore size of 50-200 μm, and the porous structure is neat and regular.
[0056] 2. Infrared absorption peak analysis was performed on the PGA-PDRN hydrogel prepared in Example 1. For example... Figure 2 As shown, at 1250-1350 cm -1 1550 -1750 cm -1 and 2900 cm -1 The absorption peaks at the wavelengths changed significantly; these are characteristic peaks of amide bonds, indicating that the PGA-PDRN crosslinking was successful.
[0057] 3. The viscoelasticity of the hydrogel was measured using a universal testing machine and a rheometer. The specific measurement results of the hydrogels prepared in Examples 1-7 and the hydrogel prepared in Comparative Example 1 are shown in Table 1.
[0058] Table 1 Performance Results
[0059]
[0060] As shown in Table 1, Examples 1-4 demonstrate that the mechanical strength of the hydrogel increases significantly with increasing PGA molecular weight. This indicates that increasing PGA molecular weight is beneficial for improving the mechanical strength of the hydrogel, possibly because larger PGA molecular chains are longer, enabling the formation of more complex and compact network structures during cross-linking, thus enhancing the hydrogel's resistance to deformation under external forces. Examples 1, 5, and 6 show that the mechanical strength of the hydrogel increases significantly with increasing PGA concentration. This suggests that increasing PGA concentration helps improve the mechanical strength of the hydrogel, possibly because a higher PGA concentration results in a greater number of molecules participating in cross-linking per unit volume, forming a denser cross-linked network, thereby improving the mechanical properties of the hydrogel. Comparative Example 1, Examples 1 and 7 show that the mechanical strength of the hydrogel increases significantly with increasing PDRN concentration. This indicates that increasing PDRN concentration has a positive impact on the mechanical strength of the hydrogel.
[0061] Furthermore, the performance data of Examples 2 and 5 are lower than those of Comparative Example 1, which does not contain PDRN. This does not indicate that PDRN is ineffective, but rather reveals that the mechanical properties of the hydrogel of this invention are fundamentally dependent on the strength of the initial network structure constructed by PGA. When the molecular weight of PGA is low or the concentration is insufficient, the initial network itself is too fragile, and even if PDRN is introduced to provide additional crosslinking, it cannot compensate for its structural defects. This indicates that the synergistic effect of PGA and PDRN provided by this invention, thereby enhancing the mechanical strength of the hydrogel, is based on an effective initial network composed of PGA with a certain molecular weight and concentration.
[0062] 4. PDRN release: The hydrogel prepared in Example 1 was placed in 500 mL of physiological saline and placed in a 37°C incubator. Samples were taken at regular intervals, and an equal amount of physiological saline was added after each sample was taken. The PDRN concentration in the sample was then measured.
[0063] PDRN concentration determination method: PDRN solutions of 0.01, 0.02, 0.03, 0.04, and 0.05 mg / mL were prepared, and their absorbance at 260 nm was measured using a UV spectrophotometer to plot a standard curve. The PDRN-containing solution samples were appropriately diluted, and their absorbance at 260 nm was measured. The PDRN concentration was calculated based on the standard curve. Note that Comparative Example 1 did not contain PDRN, therefore performance data could not be measured.
[0064] like Figure 3 As shown, PDRN exhibits a continuous and gradual release trend over a 35-day testing period. This indicates that the hydrogel prepared in this invention achieves long-term, controllable release of PDRN.
[0065] 5. Cell experiments: Proliferation experiment: Human chondrocytes were cultured at 2.5 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of 1 / 2 well in 96-well cell culture plates and incubated for 24 h. After 24 h, the cells were fully adhered to the plates. The original culture medium was removed, and the cells were washed twice with PBS buffer. Fresh culture medium containing 0.08, 0.31, 1.25, 5, 20, and 40 μg / mL hydrogel was prepared using the hydrogel from Example 1. Incubation was continued. Cell viability was assessed using the CCK-8 assay 48 h after drug addition.
[0066] like Figure 4 As shown in (a), the survival rate of human chondrocytes treated with different concentrations of hydrogel remained at a high level, with no obvious cytotoxicity; and the cell survival rate gradually increased with the increase of hydrogel concentration within a certain range. This indicates that the hydrogel prepared in this invention has excellent cell compatibility, and not only does it not inhibit the growth of human chondrocytes, but it can also effectively promote the proliferation of human chondrocytes.
[0067] Anti-inflammatory assay: RAW264.7 cells were cultured to the logarithmic growth phase, with 3.5 × 10⁶ cells per well. 4 Cells were plated into 6-well plates and cultured overnight in Medium A. Medium A was used as a blank control group; LPS (lipopolysaccharide) diluted to a final concentration of 1 μg / mL in Medium A was used as a positive control group; the experimental groups were set up as follows: LPS 1 μg / mL + 40 μg / mL hydrogel, LPS 1 μg / mL + 20 μg / mL hydrogel, and LPS 1 μg / mL + 10 μg / mL hydrogel. The solutions were added to 6-well plates and cultured for another 24 h. The cell culture supernatant was extracted and analyzed using a NO detection kit.
[0068] like Figure 4As shown in (b), the NO content in the three experimental groups was lower than that in the positive control group, and the NO content gradually decreased as the PDRN gel concentration increased. This indicates that the hydrogel prepared in this invention has an anti-inflammatory effect, and the higher the concentration, the more significant the anti-inflammatory effect.
[0069] 6. Animal Experiments: SPF-grade male SD rats aged 4-6 weeks were selected for the study. After 7 days of acclimatization, the rats underwent anterior cruciate ligament (ACLT) transection surgery to induce an osteoarthritis model. The knee joint was opened through a midline incision. The sham-operated group received no other treatment, while in the other groups, the patella was rotated laterally to expose the internal structures of the knee joint. The anterior cruciate ligament was then located and cut, followed by a drawer test to verify whether the anterior cruciate ligament was severed. The knee joint was flushed three times with physiological saline, and the wound was sutured layer by layer. Postoperatively, 400,000 units of penicillin were routinely administered intramuscularly once daily for three days. Two weeks postoperatively, the animals that underwent ACLT surgery were randomly divided into two groups: the OA group and the hydrogel group (Example 1). The OA group and the sham-operated group received physiological saline intra-articularly once a week for 5 weeks; the hydrogel group received intra-articularly once a week for 5 weeks. After euthanizing the rats, the right knee joint was exposed, and synovial fluid was extracted from the joint cavity using a syringe. The concentrations of TNF-α, IL-1β, and IL-6 in the synovial fluid were detected using an ELISA kit.
[0070] like Figure 5 As shown, in the OA group, osteoarthritis induced by ACLT surgery showed significantly elevated concentrations of three pro-inflammatory cytokines, indicating successful model establishment and severe joint inflammation. In contrast, the concentrations of the three pro-inflammatory cytokines in the synovial fluid of the hydrogel group (Example 1) were significantly lower than those in the OA group, and the concentrations of IL-1β and IL-6 were closer to those in the sham-operated group. This indicates that the hydrogel prepared in this invention can effectively inhibit local inflammatory responses in the joints of osteoarthritis model rats, reduce inflammatory factor levels, and thus alleviate osteoarthritis symptoms.
[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A hydrogel for osteoarthritis repair, characterized in that, The hydrogel is made of polydeoxyribonucleotides and polyglutamic acid, and has a porous structure with an average pore size of 50-200 μm. The hydrogel has a compressive modulus of 5-90 kPa; The molecular weight of the PGA is 106-300 kDa.
2. A method for preparing a hydrogel for osteoarthritis repair as described in claim 1, characterized in that, Includes the following steps: (1) Prepare PDRN solution and PGA solution respectively; (2) Mix the PDRN solution with the PGA solution to obtain a pre-crosslinked solution. (3) Add cross-linking agent solution to pre-cross-linking solution, stir and mix evenly, and let stand to react.
3. The preparation method according to claim 2, characterized in that, In step (1), the solvent in the PDRN solution is selected from one of PBS buffer solution, physiological saline, and deionized water, and the mass concentration of the PDRN solution is 0.01%-4%. In step (1), the solvent in the PGA solution is selected from PBS buffer solution, physiological saline, and deionized water, and the mass concentration of PGA in the PGA solution is 5%-50%.
4. The preparation method according to claim 2, characterized in that, In step (1), the crosslinking agent is selected from 1 Ethyl Carbodiimide hydrochloride, N One or more of the following: hydroxysuccinimide, 1,4-butanediol diglycidyl ether, glutaraldehyde, carbodiimide, genipin, cystamine, hexanoisocyanate, diphenylphosphine azide, and polyethylene glycol diacrylate.
5. The preparation method according to claim 2, characterized in that, In step (2), the volume ratio of the PDRN solution to the PGA solution is 1:1-3.
6. The preparation method according to claim 5, characterized in that, The volume ratio of the PDRN solution to the PGA solution is 1:
1.
7. The preparation method according to claim 2, characterized in that, In step (3), the amount of crosslinking agent solution added is 5-50 μL / ml of the pre-crosslinking solution volume.
8. The preparation method according to claim 2, characterized in that, In step (3), the reaction temperature is 20-40℃ and the reaction time is 4-12 h.
9. The preparation method according to claim 8, characterized in that, The reaction temperature is 30-40℃, and the reaction time is 6-8h.
10. The preparation method according to claim 9, characterized in that, The reaction temperature was 35°C and the reaction time was 6 hours.
11. The hydrogel of claim 1 or claim 2 The application of hydrogels prepared by any of the preparation methods described in 10 in the preparation of osteoarthritis repair drugs.