Preparation method of juvenile cartilage fragment combined light-cured lubricating coating hydrogel

Abstract

The invention relates to the technical field of joint repair materials, and discloses a preparation method of juvenile cartilage fragment combined light-cured lubricating coating hydrogel, which comprises the following steps: preparing MeHA, preparing DMA-MPC and preparing MeHA-P-DM; the invention further discloses the MeHA-P-DM hydrogel carrier and application. The hydrogel carrier MeHA-P-DM prepared by the invention can effectively deliver PJAC, has good biocompatibility, and has the advantages of improving cell survival rate and biological activity, promoting cartilage repair and regeneration, relieving inflammation and exerting good lubricating property and anti-pollution capacity; meHA (at) P (at) DM has a good transformation advantage in the aspect of PJAC implantation, and a more efficient treatment means is provided for patients suffering from joint chondropathy.

Description

Technical Field

[0001] The invention relates to the technical field of joint repair materials, and in particular to a method for preparing juvenile cartilage fragments combined with a light-cured lubricating coating hydrogel. Background Art

[0002] Articular cartilage damage caused by acute injury, trauma, and progressive degeneration can lead to severe pain, dysfunction, and limited physical activity of the joint. Clinically, due to the limited regenerative capacity of natural avascular cartilage, surgical methods such as cartilage transplantation are widely used to reconstruct damaged articular cartilage, which have the disadvantages of low healing and poor interface integration. Repair techniques including bone marrow stimulation such as drilling, microfracture, and wear arthroplasty have the advantages of being cost-effective and free of donor site complications. However, this technique results in the formation of fibrocartilage with weaker biomechanical properties than natural hyaline cartilage, which is mainly composed of type II cartilage. In addition, repair techniques such as autologous cartilage transplantation and fresh osteochondral allograft transplantation have demonstrated their success rate and can achieve subjective improvement in pain and function. Autologous cartilage transplantation involves the transfer of native extracellular matrix and viable chondrocytes in the subchondral bone from the minimal weight-bearing area of ​​the ipsilateral knee joint or, less commonly, from the anterior part of the talus to the osteochondral lesion.

[0003] Autologous cartilage transplantation can make up for the defect of low bioactivity during cartilage implantation. However, due to the lack of an ideal delivery carrier, direct implantation of autologous cartilage is still far from being widely used in clinical practice. Direct autologous cartilage transplantation may lead to a harmful inflammatory microenvironment after cartilage lesions and inevitable contamination after implantation, resulting in impaired cell viability and bioactivity. More importantly, the inevitable friction between the two joint surfaces will further induce excessive shear force, reduce cell viability, and further cause excessive inflammation, which may lead to the failure of autologous cartilage transplantation.

[0004] Recently, particulate juvenile articular cartilage (PJAC), composed of hyaline cartilage from young donors, has emerged as a novel living material for the treatment of symptomatic joint defects. PJAC exhibits superior cartilage regeneration capacity, increasing the number of chondrocytes and improving metabolic activity. Studies have shown that compared with aged chondrocytes, juvenile chondrocytes have a greater potential for genetic transformation to produce hyaline-like cartilage tissue. However, due to the fragility of PJAC, PJAC cannot be effectively delivered at present. Therefore, overcoming the defect of PJAC that cannot be effectively delivered and developing a new type of living material carrier based on this has important clinical significance for promoting cartilage regeneration. Summary of the invention

[0005] Based on the above problems, the present invention provides a method for preparing juvenile cartilage fragments combined with photocurable lubricating coating hydrogel. The present invention provides a novel hydrogel carrier system MeHA@P@DM, which can effectively deliver PJAC and has the advantages of improving cell survival rate and biological activity, promoting cartilage regeneration, reducing inflammation, and exerting good lubrication properties and anti-pollution ability.

[0006] In order to solve the above technical problems, the present invention provides a method for preparing juvenile cartilage fragments combined with a light-cured lubricating coating hydrogel, comprising the following steps:

[0007] S1: Preparation of MeHA (Methacrylated Hyaluronic Acid)

[0008] 100 mL of 2% hyaluronic acid and 10 mL of methyl acrylate were mixed to obtain a mixture, and the pH value of the mixture was adjusted to 8 using a NaOH solution. The mixture was then placed at 4° C. for reaction for 2 days to obtain an initial MeHA product. The initial MeHA product was precipitated and washed five times with ethanol, and the precipitate was collected and vacuum freeze-dried to obtain the final MeHA;

[0009] S2: Preparation of DMA-MPC (dopamine methacrylamide-2-methacryloyloxyethyl phosphorylcholine)

[0010] Dissolve 0.1 g DMA and 0.6 g MPC in 25 mL N,N-dimethylformamide solution, add 1% azobisisobutyronitrile as an initiator to the solution, and polymerize at 65°C for 36 hours to form a DMA-MPC copolymer, then collect the DMA-MPC copolymer product and dialyze for 48 hours to obtain purified DMA-MPC;

[0011] S3: Preparation of MeHA@P@DM

[0012] Granulated juvenile articular cartilage PJAC is extracted from articular cartilage, and the MeHA prepared in step S1 and the photoinitiator LAP are dissolved in a PBS solution, wherein the mass fraction of MeHA in the PBS solution is 1wt%, 2wt% or 3wt%, and the mass fraction of LAP is 0.5wt%, to obtain a prepolymer solution. The prepolymer solution is then mixed with PJAC, and PJAC is added to the prepolymer solution in an amount of 3 mg / mL, followed by irradiation under blue light for 2 minutes to form MeHA@P; MeHA@P is immersed in the DMA-MPC solution obtained in step S2 to obtain a MeHA@P@DM product.

[0013] In order to solve the above technical problems, the present invention also provides a MeHA@P@DM hydrogel carrier.

[0014] In order to solve the above technical problems, the present invention also provides the use of MeHA@P@DM hydrogel carrier in the preparation of products for cartilage treatment or regeneration.

[0015] Compared with the prior art, the beneficial effects of the present invention are: the hydrogel carrier MeHA@P@DM prepared by the present invention can effectively deliver PJAC, has good biocompatibility, and has the advantages of improving cell survival rate and biological activity, promoting cartilage repair and regeneration, reducing inflammation, and exerting good lubrication performance and anti-pollution ability; MeHA@P@DM has a good conversion advantage in PJAC implantation, providing a more efficient treatment method for patients with articular cartilage lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The characterization results of MeHA, DMC and MeHA@P@DM of the embodiments of the present invention are shown in FIG. 1 ; wherein: (A) and (B) are the 1H NMR and FTIR results of MeHA, respectively; (C) is the FTIR of DMA-MPC; (D to F) are SEM images of MeHA, MeHA@P and MeHA@P@DM hydrogels; (G to J) are the COF and comprehensive mechanical properties of MeHA, MeHA@P and MeHA@P@DM;

[0017] Figure 2 This is a photo of the cross-linking and injectability of the MeHA hydrogel according to an embodiment of the present invention;

[0018] Figure 3 Macroscopic images of PJAC hydrogels loaded with MeHA@P and MeHA@P@DM for the embodiments of the present invention;

[0019] Figure 4 The biocompatibility test results of MeHA@P and MeHA@P@DM of the embodiments of the present invention; wherein (A) is the live / dead and HE staining results of cultured MeHA@P and MeHA@P@DM; (B and C) are the cell viability and proliferation results of different hydrogels;

[0020] Figure 5 The cell migration and proliferation in MeHA@A@DM hydrogel and MeHA@P@DM hydrogel in the embodiments of the present invention; wherein: (A) is a representative image of chondrocyte migration in MeHA@P@DM and MeHA@A@DM on the 7th day of in vitro culture; (B) is the quantitative result of cell proliferation in MeHA@P@DM and MeHA@A@DM; (C) is the Transwell bottle culture of MeHA@P@DM and MeHA@A@DM; (D) is the quantification of the number of migrating cells;

[0021] Figure 6The expression of MMP14 in MeHA@P@DM and MeHA@A@DM according to the embodiment of the present invention;

[0022] Figure 7 The process of implanting hydrogel in a rabbit cartilage model for an embodiment of the present invention;

[0023] Figure 8 The following are the results of histological analysis in the embodiments of the present invention; wherein: (A) is a macroscopic photograph of repaired cartilage; (B, C) are HE and safranin o / fast green staining of repaired OC tissue; (D, E) are ICRS and histological scores and staining results of regenerated cartilage;

[0024] Fig. 9 The results of histological analysis in the examples of the present invention; wherein: (A) is the result of Col-2 immunostaining of regenerated cartilage; (B) is the quantitative result of Col-2 staining. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0026] Example:

[0027] Inspired by the chemical composition and hierarchical structure of natural cartilage, we developed a novel hydrogel carrier system, MeHA@P@DM, for the effective delivery of living materials, including PJACs with articular cartilage regeneration.

[0028] Since hyaluronic acid is the main chemical component of cartilage in nature, it has excellent biocompatibility, intrinsic regenerative ability and anti-inflammatory potential, and can support chondrocyte growth and cartilage regeneration. Therefore, the inventors first synthesized methacrylic hyaluronic acid (MeHA) from hyaluronic acid (HA) and methyl acrylate (methacrylic anhydride), using MeHA as the basic carrier material.

[0029] The preparation method of MeHA is as follows: 100 mL of 2% HA and 10 mL of methyl acrylate are mixed to obtain a mixture, the pH value of the mixture is adjusted to 8 using a NaOH solution, and then the mixture is placed at 4°C for reaction for 2 days to obtain an initial MeHA product, the initial MeHA product is precipitated and washed five times with ethanol, the precipitate is collected and vacuum freeze-dried to obtain the final MeHA.

[0030] In order to make the MeHA@P@DM hydrogel carrier have biomimetic lubrication properties, the inventors also copolymerized the adhesive DMA with the zwitterionic MPC to synthesize the adhesive lubricant DMA-MPC. The synthesis process is as follows: 0.1g DMA and 0.6g MPC were dissolved in 25mL N,N-dimethylformamide solution, and 1% azobisisobutyronitrile was added to the above solution as an initiator. After polymerization at 65°C for 36 hours, DMA-MPC copolymers were formed. After that, the DMA-MPC copolymer products were collected and dialyzed for 48 hours to obtain purified DMA-MPC.

[0031] Next, the inventors evaluated the synthesized MeHA using 1H NMR and FTIR. Figure 1 A and attached Figure 1 B, the peaks at 5.8 ppm and 6.8 ppm represent C=CH2 groups, indicating that MeHA was successfully synthesized; in addition, in the FTIR evaluation, 1640 cm -1 The characteristic peak at represents the C=C group, which also indicates that MeHA is successfully synthesized.

[0032] At the same time, the inventors also confirmed the successful synthesis of DMA-MPC through FTIR evaluation. Figure 1 C, of ​​which 1602cm -1 The characteristic peak at 1454 cm represents the C=C group of MPC. -1 and 1247cm -1 Attributed to COR (where R is the side chain of MPC), P=O, 1086cm -1 It is attributed to the stretching vibration of PO-CH2.

[0033] See attached Figure 2 After the inventors prepared MeHA solution with water (10w / v%), they found that the MeHA solution showed excellent fluidity, indicating that the MeHA solution was easy to inject during application. MeHA solution can form MeHA hydrogel after 150s of blue light cross-linking, showing its ability to be quickly implemented in critical surgeries. At the same time, the inventors simulated and verified the injectability and cross-linking of MeHA, and showed that MeHA hydrogels with different structures can be constructed.

[0034] Next, granulated juvenile articular cartilage PJAC was extracted from the articular cartilage, and the MeHA prepared above and the photoinitiator LAP were dissolved in a PBS solution, the mass fraction of MeHA in the PBS solution was 1wt%, 2wt% or 3wt%, and the mass fraction of LAP was 0.5wt%, to obtain a prepolymer solution. The prepolymer solution was then mixed with PJAC, and PJAC was added to the prepolymer solution at an amount of 3 mg / mL, followed by irradiation under blue light for 2 minutes to form MeHA@P; MeHA@P was then coated with DMA-MPC (P-DMA-MPC), and the final MeHA@P@DM sample was prepared by a simple impregnation process.

[0035] See attached Figure 3 , macroscopic images of PJAC hydrogels loaded with MeHA@P and MeHA@P@DM. For PJAC delivery vehicles, porous structures are important for maintaining cell viability and removing waste, so the inventors used scanning electron microscopy (SEM) to evaluate the microstructures of the two groups. See Appendix Figure 1 D. Attachment Figure 1 E and Attachment Figure 1 F, The inventors found that the MeHA@P group exhibited a porous structure. It is worth noting that the P-DMA-MPC coated MeHA@P@DM sample also showed a porous microstructure, which indicates that the P-DMA-MPC coating does not affect the porous microstructure of the MeHA hydrogel.

[0036] In order for the PJAC-loaded hydrogel to be successfully implanted, interfacial lubrication is crucial, as it can reduce the shear force applied to the PJAC after implantation, thereby synergistically improving cell viability. Therefore, the inventors used a universal material tester to verify the lubrication efficiency of the MeHA@P@DM hydrogel carrier. The inventors found that the MeHA and MeHA@P groups exhibited a coefficient of friction (COF) of around 3.5, which may cause significant friction between the hydrogel and nearby tissues, thereby killing the loaded cells. In addition, see Appendix Figure 1 G and Attachment Figure 1 H, the inventors found that the COF of MeHA@P@DM was significantly reduced due to the presence of the hydrated p-DMA-MPC layer. It is therefore speculated that this drastic reduction in COF can effectively mimic the lubricating microenvironment of natural cartilage, thereby improving the cell viability of implanted PJACs.

[0037] In addition to lubrication, the mechanical properties of the hydrogel are also important. If the material is too soft and cannot withstand external forces, it will collapse after implementation. Therefore, the inventors tested the mechanical properties of different groups using a mechanical tester and found that the bare HA group had the lowest mechanical properties, with a Young's modulus of 12.3 kPa. In contrast, see Appendix Figure 1 I and Attachment Figure 1The Young’s modulus of MeHA@P and MeHA@P@DM groups almost doubled. This is because the chopped PJAC can act as a reinforcing filler to increase the mechanical properties of the hydrogel.

[0038] It can be seen that the MeHA@P@DM of the present invention has excellent lubricity and mechanical properties and has great potential for cartilage regeneration.

[0039] During PJAC implantation, the viability of chondrocytes is one of the key elements for successful implementation. Therefore, the inventors evaluated the cell viability of MeHA@P and MeHA@P@DM groups by live and dead staining on days 3 and 14. Figure 4 It can be seen that the cell survival rates of the two groups on the 3rd day and the 14th day were both over 70%, and such high cell survival rates may be related to the good protective effect of HA. At the same time, it can be found that the MeHA@P and MeHA@P@DM groups had good cell proliferation on the 3rd day and the 14th day. The above results show that the hydrogel system of the present invention has good cell biocompatibility.

[0040] Next, the inventors conducted cell expansion experiments to evaluate whether the hydrogel carrier could maintain the regenerative properties of PJAC, using adult articular cartilage particle hydrogel (MeHA@A@DM) as a control group.

[0041] During long-term in vitro culture, both MeHA@P@DM and MeHA@A@DM showed that chondrocytes could migrate and proliferate in the hydrogels. Figure 5 A, It can be seen that on the 7th day, the chondrocytes in the MeHA@P@DM group migrated, while there were no cells in the MeHA@A@DM group. Figure 5 B, As the cell culture time increases, the number of proliferating cells in the MeHA@P@DM group is higher than that in the MeHA@A@DM group. Figure 5 C and attached Figure 5 D. When cultured in transwell flasks, the results were also consistent, with the MeHA@P@DM group showing increased cell migration in vertical transwell flasks. The above experimental results indicate that the MeHA@P@DM hydrogel of the present invention can effectively protect the viability of active materials in PJAC and support the migration of chondrocytes. This suggests that the MeHA@P@DM hydrogel has the potential to enhance the regenerative capacity of PJAC by preserving living cells and promoting their migration within the hydrogel matrix.

[0042] It is known that MMP14 plays an important role in cartilage remodeling and regeneration. It participates in the turnover and remodeling of the extracellular matrix during the growth and maturation of juvenile articular cartilage, ensuring the normal formation and maintenance of the tissue. To further study MMP14, we compared its expression in MeHA@P@DM and MeHA@A@DM.

[0043] See attached Figure 6 A, On day 14, immunofluorescence staining of MMP14 showed that MeHA@P@DM expressed MMP14 in both cartilage and migrating chondrocytes. Figure 6 B, Compared with the MeHA@A@DM group, the expression of MMP14 in the MeHA@P@DM group was 14 times higher. The above findings are consistent with the results of ELISA analysis of the culture medium, see Appendix Figure 6 C, it can be seen that the level of MMP14 released by the MeHA@P@DM group is higher than that of the MeHA@A@DM group. Figure 6 D, PCR results showed that chondrocytes from MeHA@P@DM showed greater cartilage regeneration potential than those from MeHA@A@DM. In summary, MeHA@P@DM can promote the expression and secretion of MMP14, which may promote cartilage remodeling and cell migration.

[0044] Next, the inventors used a rabbit cartilage defect model to characterize the therapeutic performance of the MeHA@P@DM hydrogel carrier in vivo. The implantation process is shown in the attached Figure 7 .

[0045] See attached Figure 8 The experiment was divided into blank group, MeHA group, MeHA@DM group, MeHA@A@DM group and MeHA@P@DM group. Figure 8 A, 12 weeks after surgery, gross photographs were taken to evaluate cartilage regeneration. Figure 8 D, The ICRS score of the MeHA@P@DM group was the highest, indicating good cartilage regeneration. Figure 8 B, Histological staining was used to further evaluate tissue repair. In the blank and MeHA groups, tiny new cartilage tissue formation was observed at the implantation site, which may be due to the lack of protection from the proinflammatory microenvironment or inevitable friction in these groups. In contrast, the MeHA@P@DM group showed complete repair of the defect site, the tissue was similar to natural cartilage, and was well integrated with the implant.

[0046] To verify cartilage regeneration, the inventors evaluated cartilage formation by hematoxylin-eosin (H&E) and Safranin-O / Fast-Green staining, see Appendix Figure 8C, MeHA@P and MeHA@P@DM groups both showed good cartilage repair advantages, and the defect site was filled with newly developed cartilage tissue rich in glycosaminoglycans (GAGs). Figure 8 E, HE staining and histological score analysis showed that the score of the MeHA@P@DM group was the highest among all groups.

[0047] Collagen-2 (Col-2) is a marker for cartilage repair, so the present invention also performed Col-2 staining. Fig. 9 A and attached Fig. 9 B, The MeHA@P@DM group showed the largest positive Col-2 staining area compared with the other groups. The above results collectively indicate that MeHA@P@DM has the potential to treat cartilage repair, as it exhibited better cartilage regeneration and integration in the rabbit cartilage defect model.

[0048] In summary, the inventors confirmed the successful synthesis of MeHA and p-DMA-MPC by nuclear magnetic resonance hydrogen spectrum (1H NMR) and Fourier transform infrared spectroscopy (FTIR) analysis. The MeHA solution showed excellent fluidity and could be quickly cross-linked into hydrogels by ultraviolet light; at the same time, the injectability and cross-linking of MeHA were verified, and different hydrogel structures were successfully constructed. Compared with MeHA and MeHA@P, the MeHA@P@DM carrier significantly reduced the coefficient of friction (COF) and improved the lubrication efficiency. In addition, the mechanical properties of MeHA@P and MeHA@P@DM hydrogels were enhanced, which were suitable for cartilage regeneration. The activity and proliferation of chondrocytes in the hydrogel showed good biocompatibility. In vivo studies using a rabbit cartilage defect model showed that MeHA@P@DM had a good cartilage repair effect, and the defect site was filled with newly formed cartilage tissue rich in glycosaminoglycans (GAGs). The above results prove that MeHA@P@DM can be used in the treatment of cartilage regeneration as a promising hydrogel carrier.

[0049] The above is an embodiment of the present invention. The above embodiments and the specific parameters in the embodiments are only for the purpose of clearly describing the invention verification process, and are not intended to limit the patent protection scope of the present invention. The patent protection scope of the present invention is still subject to its claims. Any equivalent structural changes made by using the contents of the description and drawings of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing juvenile cartilage fragments combined with a light-cured lubricating coating hydrogel, characterized in that: The steps include: S1: Preparation of MeHA (Methacrylated Hyaluronic Acid) S2: Preparation of DMA-MPC (dopamine methacrylamide-2-methacryloyloxyethyl phosphorylcholine) Dissolve 0.1 g DMA and 0.6 g MPC in 25 mL N,N-dimethylformamide solution, add 1% azobisisobutyronitrile as an initiator to the solution, and polymerize at 65°C for 36 hours to form a DMA-MPC copolymer, then collect the DMA-MPC copolymer product and dialyze for 48 hours to obtain purified DMA-MPC; S3: Preparation of MeHA@P@DM Granulated juvenile articular cartilage PJAC was extracted from the articular cartilage, and the MeHA prepared in step S1 and the photoinitiator LAP were dissolved in a PBS solution to obtain a prepolymer solution. The prepolymer solution was then mixed with PJAC and then irradiated under blue light for 2 minutes to form MeHA@P; MeHA@P was immersed in the DMA-MPC solution obtained in step S2 to obtain a MeHA@P@DM product.

2. The preparation method according to claim 1, characterized in that: The preparation method of methacrylated hyaluronic acid in step S1 is as follows: 100 mL of 2% hyaluronic acid and 10 mL of methyl acrylate are mixed to obtain a mixture, the pH value of the mixture is adjusted to 8 using a NaOH solution, and then the mixture is placed at 4°C for 2 days to react to obtain an initial MeHA product, the initial MeHA product is precipitated and washed five times with ethanol, the precipitate is collected and vacuum freeze-dried to obtain the final MeHA.

3. The preparation method according to claim 1, characterized in that: In step S3, the mass fraction of MeHA in the PBS solution is 1 wt %, the mass fraction of LAP is 0.5 wt %, and PJAC is added to the prepolymer solution in an amount of 3 mg / mL.

4. The preparation method according to claim 3, characterized in that: The mass fraction of MeHA in the PBS solution in step S3 is 2 wt %.

5. The preparation method according to claim 3, characterized in that: The mass fraction of MeHA in the PBS solution in step S3 is 3 wt %.

6. The MeHA@P@DM hydrogel carrier prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the MeHA@P@DM hydrogel carrier according to claim 6 in the preparation of products for cartilage treatment or regeneration.

Images