A three-dimensional biological hydrogel and its preparation method and application

Abstract

The present invention relates to the technical field of joint repair hydrogels, and in particular to a three-dimensional biohydrogel and its preparation method and application. The three-dimensional biohydrogel, by weight, comprises: 1-5 parts of methacrylic anhydride hyaluronic acid HAMA, 0.05-0.15 parts of initiator, 3-8 parts of hydrogenated soybean phosphatidylcholine liposomes and 7-13 parts of stem cell solution; the preparation method of the stem cell solution is: gelatin and water are mixed to obtain a gelatin solution; stirring at 40-60 ° C to prepare a dispersed phase; adding an emulsifier to the oil to prepare a continuous phase; the dispersed phase and the continuous phase are prepared by microfluidic technology to obtain uniform droplets, and the droplets are freeze-dried to obtain gelatin microspheres; stem cells are added to the gelatin microspheres and incubated at 25-37 ° C for 2-4 hours to obtain a stem cell solution. The present invention has long-lasting efficacy and significant safety, and can fundamentally repair articular cartilage.

Description

Technical Field

[0001] The present invention relates to the technical field of joint repair hydrogels, and in particular to a three-dimensional biohydrogel and a preparation method and application thereof. Background Art

[0002] Stem cell therapy has always been a hot topic in tissue repair and clinical treatment. In particular, stem cells have the ability to differentiate into different cell types, which gives them considerable application prospects and potential in various fields. However, precisely because of their ability to differentiate in multiple directions, if there is no suitable growth environment, stem cells are also very likely to differentiate in unexpected directions, thereby losing the expected repair and treatment effects, and may even cause side effects. Hydrogel (Hydrogel), because it is similar to the components of human tissue and has long been verified to have good biocompatibility, hydrogel scaffolds have also begun to play an important role in tissue engineering. With the exploration of the potential and market demand for stem cell therapy, by designing and constructing three-dimensional hydrogel scaffolds with specific biological microenvironments, stem cells are induced to achieve directional differentiation, growth, and proliferation, and then the repair effect is achieved in the defective tissue or lesion.

[0003] Lacuna is a unique structure of chondrocytes in natural joint tissue and three-dimensional culture (such as Figure 1 Because of this characteristic, chondrocytes tend to grow independently under three-dimensional growth conditions, forming a circular / elliptical structure with a layer of cavities, unlike the long spindle shape they exhibit when attached to a substrate during two-dimensional culture. Furthermore, according to existing literature, the radial size of the cavities is directly related to osteoarthritis (OA). Studies have shown that under OA conditions, the size of the cavities surrounding chondrocytes also increases, significantly increasing from 30-50 μm in healthy cartilage cavities to an average of 100 μm or more in OA-affected cartilage cavities ( Figure 1 A and 1B) [3] .

[0004] In addition, according to literature, when bone marrow-derived mesenchymal stem cells (BMSCs) are cultured in a three-dimensional bioscaffold with cell adhesion ligands / strong adhesion, the bioscaffold can support the mechanical conduction of the loaded cells and promote the differentiation of stem cells into osteoblasts. Conversely, in a bioscaffold without cell adhesion ligands / weak adhesion, it can support the proliferation of stem cells into spheres and the formation of a hypoxic microenvironment, making it easier for them to differentiate into chondrocytes when cultured in this independent nesting state in three-dimensional culture. [4,5]According to a paper published in Advanced Materials by Ming-Zhu Zhang's team in 2023, it was verified that when chondrocytes are cultured in three dimensions in hydrogels and are subject to the geometric constraints provided by the microenvironment, they can further promote the secretion of more extracellular matrix (ECM) by chondrocytes, such as proteoglycans and glycosaminoglycans (GAGs), and significantly inhibit the hypertrophy and dedifferentiation of chondrocytes. [3] .

[0005] Natural articular cartilage has a strong lubricating effect, and its friction coefficient COF (Coefficient of friction) can reach 0.002-0.02 [6] Currently, there are few technologies that can produce artificial materials that achieve such super-lubricity. In addition to our daily lives, the lubrication mechanism of articular cartilage also plays an indelible role in early osteoarthritis (OA) and cartilage repair. It has been documented that in early OA, the increase in COF causes chondrocytes to be stimulated by greater shear stress, thereby secreting more matrix degrading enzymes such as MMP13 and ADAMTS. [5-7] It is proposed that repairing cartilage tissue from the perspective of lubrication mechanism within the joint is a new and effective perspective and potential method. Among them, the boundary layer effect is one of the main lubrication mechanisms of natural articular cartilage. The boundary layer effect is a kind of intermolecular force that realizes the lubrication function through the "hydration shell effect" mechanism, such as Figure 2 shown.

[0006] When a highly polar central molecule appears in an aqueous solution, it attracts surrounding water molecules to form a structure similar to a "water shell". Because this structure is very stable, when the water shell is deformed by external pressure, other surrounding water molecules will immediately replenish it to maintain the stability of the structure and function of the water shell. The whole process takes about 10 -9 Seconds, so the energy loss generated is negligible, and energy loss is the most concentrated form of manifestation in the friction process. The emergence of the water shell effect can play a role in reducing energy loss in the application environment such as extrusion and wear, thereby greatly improving the lubrication effect. [8,9] .

[0007] In order to solve the problem of how to restore the super-lubricity of natural cartilage, at the end of 2020, Professor Jacob Klein's team from Israel discovered that after preparing phospholipid molecules into liposomes and then introducing them into a certain three-dimensional hydrogel scaffold, they can successfully use the boundary layer effect to make the hydrogel scaffold reach the COF of natural cartilage. This part of the work was published in the journal Science in the form of a paper.

[10] As we all know, phospholipid molecules are composed of a hydrophilic head and a hydrophobic tail. It is precisely because of this special structure that the head of phospholipids has a strong polarity and can attract the aggregation of water molecules. Liposomes are spherical molecules (such as nanometer-scale) formed by phospholipids through microfluidics, extrusion or ultrasound. Figure 3 As shown in A), because its outer layer is all hydrophilic structure, and the hydrophobic end is wrapped in the ball, the hydrophilicity and polarity of the liposome are greatly enhanced. Liposomes themselves can be divided into multilamellar large vesicles (MLV), large unilamellar vesicles (LUV), and small unilamellar vesicles (SUV) according to their particle size (as shown in Figure 3 B) and other types, and have a larger specific surface area, which can further enhance polarity and hydrophilicity.

[0008] It is precisely because of this epoch-making theoretical and experimental basis that super-lubricating hydrogels are full of prospects in the application of cartilage repair and clinical treatment.

[0009] However, the current commercial products and technologies for cartilage repair mainly include:

[0010] 1.AqueousJoint [1] :

[0011] AqueousJoint is a product launched by Liposphere. It utilizes boundary layer lubrication as a lubrication mechanism, creating an ultra-low friction interface within joints and acting as a boundary lubricant. It claims to possess unique biolubricating properties, demonstrating significant efficacy in primary synovial joints subjected to high pressure and heavy loads.

[0012] Application:

[0013] This product is mainly injected into the joint cavity to achieve the effect of relieving pain and preventing it.

[0014] Possible problems (shortcomings):

[0015] 1) Application method:

[0016] The traditional injection method is used, which is prone to inaccurate injection position; the traditional injection method also has the risk of pain, swelling and fluid accumulation;

[0017] 2) Potential risks are unknown:

[0018] This product only passed clinical review at the end of 2022 and has just been launched on the market. There is little market feedback, and whether there are any potential risks remains unknown;

[0019] 3) High price;

[0020] 4) Product effect:

[0021] The current positioning of this product is relatively vague, and it is mainly focused on preventing and relieving joint pain. Whether it can completely repair and treat joint diseases such as joint defects and osteoarthritis, the effect and duration are still unknown.

[0022] 2.Durolane [2] :

[0023] Durolane is a single-injection hyaluronic acid (HA) treatment indicated for the relief of pain associated with knee osteoarthritis and for providing joint lubrication.

[0024] Durolane's mechanism of action is primarily based on hyaluronic acid (HA) injections and involves multiple factors, such as mechanical lubrication and protection of joints, covering sensory nerve endings, binding to molecules of pain mediators, and possibly playing a nutritional metabolic role within joints.

[0025] 1) Application method:

[0026] As with the former, it is injected into the articular cavity.

[0027] 2) Possible problems (shortcomings):

[0028] Common side effects include pain, swelling, warmth, redness, and / or fluid accumulation at the injection site. Serious side effects, while rare, may include allergic reactions, infections, and bleeding into the joints.

[0029] Research has found that the half-life of this product in the rabbit knee joint is about 32 days. There is currently a lack of detailed data on its effect in humans (which also depends on individual differences). If rabbit samples are used as a reference, the product's duration is relatively short and long-term injections are required, which will cause inconvenience to customers' lives and also bring a greater economic burden.

[0030] 3. Platelet-Rich Plasma (PRP) therapy:

[0031] Platelet-rich plasma (PRP) therapy is a form of regenerative medicine that uses components of a patient's own blood to promote healing, particularly in damaged tissues such as cartilage. However, PRP is not sold as a branded "product" like a drug or medical device; rather, it is a procedure performed in a clinical setting. The process involves drawing a sample of the patient's blood, concentrating it through centrifugation to concentrate platelets and growth factors, and then injecting this concentrated mixture into the injured area to promote healing and repair. Currently, several biomedical companies have the technology to provide PRP therapy.

[0032] 1) Principle:

[0033] PRP primarily uses the patient / client's own blood components to repair and treat joints, which can significantly reduce inflammatory responses.

[0034] 2) Application method:

[0035] The injection method is similar, but the preparation process requires blood to be drawn from the patient, processed to concentrate the platelets, and then injected.

[0036] 3) Possible problems (shortcomings):

[0037] Because it's derived from the patient's own blood, side effects are generally fewer, but the risks associated with the injection still exist.

[0038] Looking at the existing technologies and products, it is not difficult to find that most of them currently use intra-articular injections to improve the intra-articular environment through drug products / autologous plasma, thereby preventing and relieving pain. Although this type of effect is relatively conservative and simple to operate, it cannot achieve precise positioning and is prone to side effects such as pain and swelling at the injection site. In addition, it is well known that articular cartilage itself does not have the ability to repair itself, and most of the above products only have a temporary effect of relieving pain and improving the internal environment. The root cause of the disease always exists and cannot be completely eliminated. Moreover, most drugs exist in the joint cavity in the form of liquids, which have poor stability and cannot guarantee long-term effectiveness. Customers / patients need to receive long-term regular injections to maintain their effectiveness, which seriously affects their quality of life and also creates great economic pressure.

[0039] Prior art CN114146226A discloses a bone marrow mesenchymal stem cell / degradable hydrogel composite material and its preparation method and use. This material uses polyethylene glycol derivatives and active oxygen responsive small molecule diamine as raw materials to prepare a polyethylene glycol hyperbranched polymer containing active oxygen responsiveness as a component of the hydrogel, hyaluronic acid modified with methacrylate as another component, cell adhesion peptides, basic fibroblast growth factor and epidermal growth factor as bioactive ingredients, and bone marrow mesenchymal stem cells are loaded therein, and prepared by a method of cross-linking gelation induced by ultraviolet radiation. However, this prior art introduces cell adhesion peptides, which causes mesenchymal stem cells to tend to grow in attachment, which is completely different from the independent / suspended growth mode of cartilage differentiation and cannot be used for cartilage differentiation applications.

[0040] Prior art CN115501253A discloses a method for preparing a combined stem cell and hydrogel biomaterial and its application in spinal cord injury, characterized in that it comprises the following steps: preparing methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA); preparing a hydrogel solution, and encapsulating standard cultured mesenchymal stem cells and / or neuroepithelial stem cells in the prepared hydrogel to obtain stem cell-loaded hydrogel or hydrogel microspheres; transplanting the stem cell-loaded hydrogel or hydrogel microspheres into the spinal cord injury for treatment and evaluating the effect. This invention is used to repair spinal cord injury, uses biomaterials with low immunogenicity, good cell biocompatibility, and certain biological functions; after transplantation, it can reduce the excessive aggregation of astrocytes after spinal cord injury, reduce the formation of glial scars, improve bladder and motor function, promote the regeneration and repair of nerves after spinal cord injury, and thus promote behavioral recovery in animals with spinal cord injury.

[0041] Prior art CN118178734B discloses a preparation method and application of a 3D stem cell-loaded composite hydrogel, belonging to the field of wound repair hydrogel technology. The preparation method includes: S1, preparing GelMA micro-hydrogel using a microfluidic method; S2, preparing an aqueous solution with a total mass concentration of HAMA and GelMA of 3wt%-8wt%, adding 10-100 freeze-dried GelMA micro-hydrogels to the aqueous solution of HAMA and GelMA, adding 0.5wt%-3wt% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and shaking to obtain a composite solution; S3, taking 100μL-300μL of a 1×10 6 mL -1 -5×106mL -1The stem cell suspension is added to the composite solution obtained in step S2 and shaken to mix thoroughly. The solution is transferred to a silicone mold and photocured to form a 3D stem cell-loaded composite hydrogel. This invention describes a method for preparing and applying a 3D stem cell-loaded composite hydrogel. GelMA microhydrogels and stem cells are loaded into a HAMA-GelMA hydrogel, and the growth of stem cells within the 3D hydrogel environment is regulated by the degradation of the GelMA microhydrogel. However, this prior art is intended for skull repair, specifically osteoblast repair.

[0042] Both of the above technologies use GelMA micro-hydrogels and participate in the three-dimensional culture of stem cells and the entire repair process. GelMA is called methacrylic anhydride gelatin, which is modified from natural gelatin (Gelatin).

[11] In GelMA, stem cells tend to adhere to the wall, promoting their differentiation into osteoblasts or neurons. However, adherent growth is not conducive to chondrogenic differentiation and cartilage repair. Therefore, the above two existing technologies are not suitable for cartilage repair.

[0043] References:

[0044] 1.Liposphere.Company-Liposphere.Available from:https: / / www.lipo-sphere.com.

[0045] 2.DUROLANE.DUROLANE-Durolane.Available from:https: / / durolane.com / en / durolane / .

[0046] 3.Ding, SL, et al., Cartilage Lacuna-Inspired Microcarriers DriveHyaline Neocartilage Regeneration. Adv Mater, 2023.35(30):p.e2212114.

[0047] 4. Yang, B., et al., Enhanced mechanosensing of cells in synthetic 3D matrix with controlled biophysical dynamics. Nature Communications, 2021.12(1):p.3514.

[0048] 5.Yang,X.,et al.,Coacervation-Mediated Cytocompatible Formation ofSupramolecular Hydrogels with Self-Evolving Macropores for 3D MulticellularSpheroid Culture.Adv Mater,2023.35(24):p.e2300636.

[0049] 6.Radin,E.L.,D.A.Swann,and P.A.Weisser,Separation of a hyaluronate-free lubricating fraction from synovial fluid.Nature,1970.228(5269):p.377-8.

[0050] 7.Lin,W.and J.Klein,Recent Progress in Cartilage Lubrication.AdvMater,2021.33(18):p.e2005513.

[0051] 8.Goldberg,R.,et al.,Boundary lubricants with exceptionally lowfriction coefficients based on 2D close-packed phosphatidylcholineliposomes.Adv Mater,2011.23(31):p.3517-21.

[0052] 9.Jahn,S.,J.Seror,and J.Klein,Lubrication of Articular Cartilage.AnnuRev Biomed Eng,2016.18:p.235-58.

[0053] 10.Lin,W.,et al.,Cartilage-inspired,lipid-based boundary-lubricatedhydrogels.Science,2020.370(6514):p.335-338.

[0054] 11.Van Den Bulcke,AI,Bogdanov,B.,De Rooze,N.,Schacht,EH,Cornelissen,M.,&Berghmans,H.(2000).Structural and rheological properties ofmethacrylamide modified gelatin hydrogels.Biomacromolecules,1(1),31-38.https: / / doi.org / 10.1021 / bm990017d Summary of the Invention

[0055] The purpose of the present invention is to provide a three-dimensional biohydrogel that can fundamentally repair and heal articular cartilage, as well as a preparation method and application thereof.

[0056] In order to achieve the above object, the technical solution of the present invention is as follows:

[0057] A three-dimensional biohydrogel, whose raw materials include, by weight: 1-5 parts of methacrylic anhydride hyaluronic acid HAMA, 0.05-0.15 parts of initiator, 3-8 parts of hydrogenated soybean phosphatidylcholine liposomes and 7-13 parts of stem cell solution;

[0058] The preparation method of the stem cell solution comprises the following steps: mixing gelatin and water to prepare a gelatin solution; stirring the mixture at 40-60° C. to prepare a dispersed phase; adding an emulsifier to the oil to prepare a continuous phase; preparing the dispersed phase and the continuous phase into uniform droplets using microfluidics technology; freeze-drying the droplets to obtain gelatin microspheres; and adding 5*10 5 / mL of stem cells and incubate at 25-37°C for 2-4 hours to obtain a stem cell solution.

[0059] The density of stem cells was calculated based on the volume of the stem cell solution.

[0060] Methacryloylated hyaluronic acid (HAMA) serves as the main component of the hydrogel scaffold; hydrogenated soybean phosphatidylcholine (HSPC) liposomes serve as an important lubricating unit in the hydrogel scaffold; gelatin microspheres adhere to cells in the three-dimensional scaffold and degrade into artificial cavities during the subsequent cultivation process, restoring the growth environment of natural chondrocytes.

[0061] In the present invention, the three-dimensional biohydrogel is attached to the joint defect or the patient's place. In the early stage, the high biocompatibility and biological fit of the hydrogel are used to achieve the effect of artificial natural articular cartilage, which can relieve the patient's pain and inhibit the deepening of the disease. In the middle and late stages, because the three-dimensional hydrogel scaffold provides a very favorable growth and differentiation environment for stem cells, when the scaffold gradually degrades with the incubation of cells, the stem cells also differentiate into chondrocytes and secrete a rich cytoplasmic extracellular matrix to fill the joint defect, thereby playing a role in repairing and healing articular cartilage from the root. The present invention can restore the growth microenvironment of chondrocytes, thereby further promoting the differentiation of mesenchymal stem cells towards the cartilage phase, and secreting more cartilage extracellular matrix, such as type II collagen and polysaccharide protein, etc. in the subsequent growth process, thereby improving the repair ability of articular cartilage.

[0062] In a preferred embodiment, the stem cells are any one of mesenchymal stem cells and induced pluripotent stem cells.

[0063] In one preferred embodiment, the mass concentration of gelatin in the gelatin solution is 8%-15%.

[0064] In one preferred embodiment, the oil is mineral oil or vegetable oil.

[0065] In a preferred embodiment, the emulsifier is Tween 80.

[0066] In one preferred embodiment, the mass ratio of the oil to the emulsifier is 95-99:1-5.

[0067] In one preferred embodiment, the volume ratio of the continuous phase to the dispersed phase is 5-10:1.

[0068] In one preferred embodiment, in the process of preparing uniform droplets by microfluidic technology, the flow rate of the dispersed phase is 30-40 μL / min; the flow rate of the continuous phase is 500-600 μL / min.

[0069] Too high or too low a flow rate will result in uneven and unstable droplet sizes.

[0070] In one preferred embodiment, the droplets are freeze-dried and then washed with ethanol or water before freeze-drying.

[0071] In one preferred embodiment, the freeze-drying temperature is -40 to -20°C, the vacuum degree is below 200 Pa, and the time is 18-24 hours.

[0072] In one preferred embodiment, the particle size of the gelatin microspheres is less than 65 μm.

[0073] The gelatin microspheres prepared by the microfluidic method of the present invention can have their diameter controlled to be less than 65 μm, and the size of the "artificial cavity" left after the gelatin degradation is adapted to the growth microenvironment of normal chondrocytes; 60 μm is a cavity size that is very close to and fits normal chondrocytes. If the particle size of the gelatin microspheres is too large, the cavity formed subsequently will be closer to the cavity size of hypertrophic chondrocytes (chondrocytes with OA lesions), which can easily restore the cell microenvironment of the lesion or inflammatory state. The present invention uses gelatin as a hydrogel to form a microsphere morphology through a process, and controls it to an ideal size that fits normal chondrocytes. On the other hand, the gelatin microspheres of the present invention will degrade rapidly at physiological temperature, leaving an "artificial cavity" suitable for stem cells to differentiate into the cartilage phase in the early stage.

[0074] In one preferred embodiment, the particle size of the gelatin microspheres is 40-60 μm.

[0075] Based on the same inventive concept, the present invention also claims a method for preparing the three-dimensional biohydrogel, comprising the following steps:

[0076] S1. adding an initiator to methacrylic anhydride-modified hyaluronic acid (HAMA) to obtain a hydrogel precursor solution;

[0077] S2. Add hydrogenated soybean phosphatidylcholine liposomes and stem cell solution to the hydrogel precursor solution, mix them evenly, and obtain a composite solution; transfer the solution to a mold and form a three-dimensional biohydrogel by photocuring.

[0078] In one preferred embodiment, the preparation method of the methacrylic anhydride hyaluronic acid HAMA is:

[0079] In a dark environment, equal masses of methyl methacrylate and hyaluronic acid are mixed evenly on ice, reacted for 10-14 hours, and the pH is maintained at 8-8.5. The obtained solution is dialyzed and freeze-dried to obtain methacrylic anhydride hyaluronic acid HAMA.

[0080] In a preferred embodiment, the molecular weight of the hyaluronic acid is 100,000-200,000.

[0081] In one preferred embodiment, the dialysis process is: transferring the solution into a dialysis bag with a molecular weight cut-off of no more than 20 kDa, and dialysis in water for 1-3 days.

[0082] In a preferred embodiment, hydrogenated soybean phosphatidylcholine liposomes are first dispersed and then added dropwise to the hydrogel precursor solution.

[0083] In one preferred embodiment, the process of dispersing hydrogenated soybean phosphatidylcholine liposomes is: ultrasonically dispersing hydrogenated soybean phosphatidylcholine liposomes in water, with an ultrasonic frequency of 600-700W and an ultrasonic time of 10-20min, to disperse hydrogenated soybean phosphatidylcholine liposomes.

[0084] In one preferred embodiment, the light curing is carried out at 365 nm, 8 mW / cm 2 -12mW / cm 2 Curing under blue light for 4-8 minutes.

[0085] In one preferred embodiment, the mold is obtained by performing a CT scan on the patient's articular cartilage defect, and the scan results are imported into a 3D bio-printing machine to generate a corresponding printing model and parameters to obtain a mold.

[0086] Based on the same inventive concept, the present invention also claims protection for the use of the three-dimensional biohydrogel in preparing a preparation for cartilage repair.

[0087] In the present invention, nano-scale liposomes are constructed and implanted into a three-dimensional hydrogel scaffold. This significantly enhances the lubricity of the scaffold, approaching or even reaching the COF of natural cartilage (approximately 0.002-0.02). Physiologically, when cells are cultured three-dimensionally in the super-lubricating hydrogel, the superior lubricity reduces shear stress stimulation experienced by the cells, thereby downregulating the gene expression and protein secretion of MMP13, ADAMTS 5, and type X collagen (Col X), which contribute to cell hypertrophy and matrix degradation. Furthermore, because the three-dimensional environment more closely resembles the superficial layer of natural articular cartilage, the expression of the proteoglycan 4 (PRG4) gene, which is linked to lubricin, is upregulated in chondrocytes, leading to the secretion of lubricin, a chemical substance in the matrix that has a lubricating effect. This further enhances the biological microenvironment of cartilage, achieving the desired cartilage repair and clinical therapeutic effects.

[0088] According to some basic cell and animal experimental data we have, Figure 4-9), when cells were cultured in the super-lubricating hydrogel we constructed in three dimensions, they not only showed high biocompatibility, but also, compared with the material control group, the various cartilage-forming indicators of the cells in the super-lubricating hydrogel were significantly improved, such as secreting more extracytoplasmic matrix (ECM), and the repair effect was more obvious in the osteochondral defect model (Osteochondral defect model), not only significantly increasing the expression of PRG4 in the superficial area, but also generating more type II collagen instead of type I collagen, which means that the cells repaired in the scaffold formed more hyaline cartilage instead of fibrous cartilage.

[0089] Therefore, the mechanism of action of the present invention is: taking osteoarthritis (OA) as an example, its main feature is that the structural degeneration and disorder of the extracellular matrix cause the lubrication effect in the joint to decline, friction increases, and then the stress-stimulated healthy chondrocytes appear hypertrophic characteristics, and the hypertrophic chondrocytes further secrete matrix enzymes (such as MMP13 and ADAMTS5), deepen the destruction of the joint environment, thereby forming a vicious cycle; coupled with the fact that articular cartilage itself does not have the ability to repair, so the consequences will only be unimaginable. The present invention, by introducing super-lubricating hydrogel in defect and diseased area, first plays the role of a buffer zone, alleviates the stress stimulation of the hypertrophic chondrocytes in the lesion, thereby suppressing its production of more matrix enzymes; meanwhile, the stem cells in the hydrogel scaffold gradually differentiate into healthy chondrocytes, and secrete abundant chondrocyte extracellular matrix (such as type II collagen and proteoglycans ...), on this basis, the original hydrogel scaffold will degrade in vivo thereupon, and the degradation product (hyaluronic acid) is exactly one of the important components in the environment of articular cartilage, and the product produced by the cells in the scaffold is exposed, reaching the effect of filling and repairing the defective structure in the lesion.

[0090] The present invention has the following beneficial effects:

[0091] Current products and treatment technologies on the market are generally conservative, focusing primarily on relieving symptoms and temporarily improving joint function, and struggle to fundamentally address the challenge of articular cartilage repair. Existing technologies are limited in their therapeutic efficacy, often failing to effectively achieve long-term repair and preventing subsequent cartilage degeneration and recurrence. The stem cell-based ultra-lubricating three-dimensional biohydrogel proposed in this invention can fundamentally repair defects and lesions in articular cartilage, with significant clinical significance.

[0092] Compared with the existing technology, the present invention not only has excellent therapeutic effects, but also is relatively simple in design and manufacturing process, and the materials and reagents used are highly biocompatible. These materials can be safely degraded in the body and will not cause adverse reactions such as immune rejection and inflammation, ensuring the safety of the treatment process. Figure 8 and Figure 9 As shown, the present invention is based on the existing basic experimental data and verifies that the biological scaffold has excellent biocompatibility in animals (mouse model). In the osteochondral defect model, the scaffold can fit closely to the defect boundary, while promoting the accelerated growth of cells in the scaffold, and promoting the up-regulated expression of extracellular matrix (ECM) and lubricin (PRG4). At 4 weeks after surgery, the mouse osteochondral defect model has shown a significant repair effect; in the longer 8 weeks and 12 weeks, the repair effect is more obvious and lasting. At the same time, the super-lubricating hydrogel bioscaffold produces more type II collagen rather than type I collagen, which means that the cells in the scaffold produce more hyaline cartilage rather than fibrous cartilage.

[0093] Compared with existing therapeutic products on the market, although drug injection can quickly relieve pain and achieve therapeutic effects in the short term, due to the instability of the injection target and the gradual degradation of the drug, its effect will quickly decay after reaching the peak, and the patient will soon return to the state before treatment, so it is necessary to repeatedly use this type of product to maintain a certain therapeutic effect. In contrast, the clinical efficacy of the present invention shows a trend closer to the change of an exponential function. With the gradual degradation of the stent in the body and the gradual increase in the secretion of matrix by cells, it can not only effectively relieve the patient's pain, but also fundamentally repair the lesion site, achieving the effect of long-term healing. This feature makes the present invention far superior to existing market products in terms of therapeutic effect, with more lasting and stable clinical performance.

[0094] In general, the beneficial effects of the present invention are:

[0095] (1) Long-lasting efficacy: The course of treatment for drugs that prevent and relieve pain in patients / clients through intra-articular injection is short, and the effect needs to be maintained through regular injection of the product. The present invention has corresponding effects at different stages of the entire efficacy. For example, during early implantation, its super-lubricating effect can effectively reduce the stress stimulation of chondrocytes at the lesion site and inhibit the production of more matrix enzymes; in the middle and late stages, the stem cells in the scaffold gradually differentiate into chondrocytes and begin to secrete chondrocyte matrix. As the original scaffold gradually degrades, more growth space is freed up for the newly differentiated chondrocytes and the newly secreted matrix, which not only fills the defect at the lesion site, but also has mechanical properties that match the level of natural cartilage, thereby restoring the articular cartilage to its original state. Therefore, the main advantage of the present invention is that it can fundamentally and thoroughly solve the problem at one time, and its repair effect will become more and more obvious as time goes by, until the properties of natural cartilage are restored.

[0096] (2) Commercialization: The materials used in the present invention are all approved by the FDA and have excellent biocompatibility. They can be used in biological experiments and clinical applications without causing immune rejection or inflammatory reactions in the human body, and the procurement cost is relatively low. The processes involved, such as microfluidics, extrusion molding, ultrasonic fragmentation, or 3D bioprinting molding technology that may be involved, all have relatively mature systems in the current industrial market. Therefore, the product preparation cost of the present invention is low, the process is mature, and large-scale commercial production can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 Figure 1: The effect of OA on the size of cavities surrounding chondrocytes. A shows the morphology of cavities in healthy and osteoarthritis (OA) cartilage tissues. B shows the quantitative analysis of the radial size of cavities in healthy and OA-affected joints.

[0098] Figure 2 Schematic diagram of the actual composition and application of superlubricating hydrogel scaffold and boundary layer lubrication effect (water shell effect);

[0099] Figure 3 is the structure of phospholipid molecules; A is a schematic diagram of the three-dimensional structure of liposomes; B is a schematic diagram of the three different types of liposomes divided into SUV, LUV, and MLV according to particle size;

[0100] Figure 4 The friction performance of three-dimensional biohydrogel scaffolds containing different concentrations of liposomes (0-100mM) was tested using a mechanical properties testing instrument and verified that the lubrication effect was significantly improved. A shows the lateral force (friction force) and normal stress between each group, which were converted into COF (coefficient of friction) as shown in the figure. B shows the relationship between the statistically obtained COF and the concentration of liposomes mixed in the bioscaffold.

[0101] Figure 5 The Young's modulus of three-dimensional biohydrogel scaffolds containing different liposome concentrations (0-100 mM) was measured using a mechanical properties testing instrument. The results confirmed that the hardness of the hydrogel scaffolds was close to that of the chondrocyte matrix, making it suitable for cell growth. A is the stress-strain curve obtained by recording stress and strain; B is the relationship between the statistically obtained Young's modulus and the concentration of liposomes mixed in the bioscaffold.

[0102] Figure 6 To transplant cells into the super-lubricating biohydrogel scaffold, the cells maintained a high survival rate (greater than 85%) after 7 days of in vitro culture at various liposome concentrations (0-100mM);

[0103] Figure 7To transplant cells into the super-lubricating biohydrogel scaffold, cells secreted more cartilage extraplasmic matrix in the high liposome concentration group;

[0104] Figure 8 To establish an osteochondral defect model in mice, a control group of cells and an experimental group of super-lubricating hydrogel scaffolds were injected into the defect. After 8 weeks of observation, it was found that the experimental group had significantly better repair effects at the defect site than the control group. A shows hematoxylin-eosin (H&E) staining, which verifies that the experimental group adhered well to the native tissue and exhibited a significant repair effect. B shows Sudan red (Safranin-O) staining, which verifies that the cells in the experimental group secreted more cartilage extracellular matrix, and overall chondrogenic differentiation was more obvious.

[0105] Figure 9 Immunohistochemical staining of PRG4 was performed in an 8-week mouse osteochondral defect model to detect the effect of lubrication on cells in the experimental group. A shows that the expression of PRG4 in the superficial layer of the experimental group was significantly higher than that in the defect and material control groups, and was close to the expression level in the SHAM group. B shows the negative control group without incubation with PRG4 antibody. DETAILED DESCRIPTION

[0106] In order to make the purpose, technical solutions and advantages disclosed in the embodiments of the present invention more clear, the embodiments of the present invention are further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0107] Example 1

[0108] Preparation of macroscopic HAMA hydrogel scaffolds

[0109] 1g of sodium hyaluronate (HA) (molecular weight 100,000-200,000, Catalog No. H-0443571, HEOWNS, Tianjin, China) was dissolved in 100mL of aqueous solution. Once fully dissolved, 1mL of methacrylic acid (Catalog No. M-58350, HEOWNS, Tianjin, China) was added. The solution was then incubated at 4°C and maintained at a pH of 8-8.5 for 12 hours. After full reaction, the solution was dialyzed through dialysis tape (8000-14000 kDa). After two days, dialysis was complete and the solution was freeze-dried to obtain HAMA powder.

[0110] The obtained HAMA powder was dissolved in PBS solution at 3% (w / v). A 0.1% (w / v) photoinitiator (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) (Cat. No. H137984, Aladdin, China) was dissolved in the above solution to form a HAMA hydrogel precursor solution. The solution was mixed by vortexing and sonication, and then the solution was stirred at a wavelength of 365 nm and 10 mW / cm 2 The HAMA hydrogel scaffold was formed by irradiating and curing under blue light for 5 minutes.

[0111] Example 2

[0112] Preparation of gelatin microspheres

[0113] Gelatin (300 bloom, type A, Sigma-Aldrich, USA) was dissolved in preheated distilled water (40-60°C) and then dissolved in PBS at a 10% (w / v) concentration to prepare a gelatin solution. Stirring was performed until completely dissolved, maintaining the solution temperature at 40-60°C to prevent gelatin coagulation. An appropriate amount of mineral or vegetable oil was added with an emulsifier (Span 80) and stirred until homogenized. This served as the continuous phase (emulsifier concentration was 2% by volume). The gelatin solution was injected into the dispersed phase channel of the microfluidic chip via a syringe pump at a controlled flow rate of 30 μL / min. The continuous phase was injected into the continuous phase channel of the chip via another syringe pump at a controlled flow rate of 500 μL / min. The volume of the continuous phase was 10 times that of the dispersed phase, ensuring that the resulting gelatin microspheres had a particle size of approximately 50 μm. With these parameters in place, the gelatin solution formed uniform droplets (precursor to gelatin microspheres) within the microfluidic chip under the shearing action of the oil phase. The formed gelatin droplets are collected in a container containing the oil phase and maintained at a low temperature (4°C) to prevent droplet fusion. In order to solidify the gelatin microspheres, the temperature is maintained at 4°C for a period of time. After the solidification reaction is sufficient, the microspheres are washed with ethanol multiple times to remove excess oil phase. Finally, the microspheres are rinsed with distilled water and freeze-dried. The freeze-drying temperature is -40 to -20°C, the vacuum degree is below 200Pa, and the time is 20 hours to obtain dry gelatin microsphere powder.

[0114] Example 3

[0115] Preparation of nano-sized liposomes

[0116] Hydrogenated soybean phosphatidylcholine (HSPC) (Lipoid, GmbH, Germany) was dissolved in PBS to obtain a 100 mM HSPC phospholipid solution. The phospholipid solution was then ultrasonically reacted under certain ultrasonic conditions (600 W, 15 min). After the ultrasonic reaction was completed, the prepared HSPC liposome suspension was obtained.

[0117] Sucrose is added to the prepared HSPC liposome suspension to a concentration of 5-10% (w / v) (the purpose of adding sucrose here is to protect the liposomes during the subsequent freeze-drying process). After mixing evenly, the liposome-sugar mixed solution is rapidly frozen. After rapid freezing, the mixture is placed in a freeze dryer to obtain a freeze-dried powder of the HSPC liposomes.

[0118] Example 4

[0119] Construction of HAMA / Gel-lipo hydrogel scaffold

[0120] The HAMA lyophilized powder obtained above was dissolved in PBS at a concentration of 3% (w / v) to obtain a HAMA hydrogel presolution. Subsequently, lyophilized gelatin microspheres were added at a concentration of 10% (w / v). HSPC liposome powder was dissolved in the HAMA hydrogel presolution at concentrations of 0, 10 mM, 50 mM, and 100 mM, respectively. 0.1% (w / v) of a photoinitiator (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) was dissolved in the mixed solution, and the mixture was then mixed uniformly by vortexing and sonication to obtain HAMA / Gel-lipo mixtures containing four different concentrations of HSPC liposomes.

[0121] After mixing evenly, the obtained HAMA / Gel-lipo mixture was dripped into the mold and heated at a wavelength of 365nm, 10mW / cm 2 The HAMA / Gel-lipo hydrogel scaffold was formed by irradiating and curing under blue light for 5 minutes.

[0122] The friction performance of the HAMA / Gel-lipo hydrogel scaffold was tested using a mechanical properties testing instrument, Mach-1 (Biomomentum, Canada). Using PBS as the liquid environment and a glass slide as the substrate, the hydrogel was repeatedly subjected to lateral displacement (20 mm, 1 mm / s, 3 repetitions). The friction force (Ff) and normal stress (FN) generated by the hydrogel scaffold during displacement were recorded using a mechanical sensor above. The results were then calculated using the following formula:

[0123] Friction coefficient (μ) = (friction force (F_f)) / (normal stress (F_N));

[0124] The friction coefficient (μ) was used to characterize the lubrication performance of the hydrogel scaffold in a simulated biological fluid environment.

[0125] The results are as follows Figure 4As shown, the friction performance of three-dimensional biohydrogel scaffolds containing different concentrations of liposomes (10mM, 50mM and 100mM) was tested by a mechanical performance testing instrument and verified that the lubrication effect was significantly improved; (A) By recording the lateral force (friction force) and normal stress between each group, and converting them into COF (coefficient of friction) as shown in the figure; (B) by statistically analyzing the relationship between the COF and the concentration of liposomes mixed into the bioscaffold. It shows that when the present invention introduces liposomes into the bioscaffold, the lubrication effect of the entire scaffold is significantly improved, especially in the high concentration groups (50mM and 100mM), the lubrication coefficient of both groups is significantly reduced compared with the control group, and the COF is close to the range of 0.003-0.02, which is very close to the level of natural cartilage (the COF of natural human articular cartilage is in the range of 0.003-0.02.).

[0126] The Young's modulus of the HAMA / Gel-lipo hydrogel scaffold was tested using a Mach-1 (Biomomentum, Canada) mechanical testing instrument in compressive mode. A glass slide was used as the substrate. The downward displacement of the hydrogel under this condition (lateral displacement: 80% of the hydrogel scaffold height, speed: 0.0167 mm / s, number of repetitions: 3) was recorded. The stress (σ) generated by the hydrogel scaffold during this displacement was recorded by a force sensor above. The stress was then calculated using the formula:

[0127] Young's modulus (E) = (stress (σ)) / (strain (ε));

[0128] Young's modulus (E) was used to characterize the hardness properties of the hydrogel scaffold in a simulated biological microenvironment.

[0129] The results are as follows Figure 5 As shown, the Young's modulus of three-dimensional biohydrogel scaffolds containing different concentrations of liposomes (0-100mM) was tested by a mechanical properties testing instrument and verified that the scaffold hardness was maintained in a relatively stable range (10-12kPa); (A) By statistically analyzing the relationship between the Young's modulus and the concentration of liposomes mixed in the bioscaffold, it can be seen that the Young's modulus data of the high concentration groups (50mM and 100mM) were significantly lower than those of the control group.

[0130] Example 5

[0131] Construction of HAMA / Gel-lipo cell / hydrogel scaffold

[0132] Before mixing stem cells, HAMA, gelatin microspheres, and nanoliposomes, ensure that all reagents and procedures in the following steps are performed under sterile conditions.

[0133] A certain amount of mesenchymal stem cells (MSCs) were prepared. 10% (w / v) gelatin microspheres and a cell suspension were thoroughly mixed at a density of 500,000 cells / mL. The mixture was incubated at room temperature (25°C) for 4 hours to allow the cells to adhere to the gelatin microspheres. After the adhesion process was complete, HAMA was dissolved in the cell / gelatin microsphere mixture at a concentration of 3% (w / v). HSPC freeze-dried powder was dissolved in the cell / gelatin microsphere mixture at concentrations of 0, 10mM, 50mM, and 100mM, respectively. A photoinitiator (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) was dissolved in the mixture at 0.1% (w / v). The mixture was then mixed thoroughly by vortexing and sonication to obtain a HAMA / gel-lipo / cell mixture.

[0134] After mixing evenly, the obtained HAMA / Gel-lipo / cell mixture was dripped into the mold and heated at a wavelength of 365nm, 10mW / cm 2 The HAMA / Gel-lipo cell / hydrogel scaffold was formed by irradiating and curing under blue light for 5 minutes.

[0135] HAMA / Gel-lipo cells / hydrogel scaffolds were cultured at 37°C in a 5% CO2 environment. The culture medium was replaced every two days. On the 7th day of in vitro culture, the cells were tested using a cytotoxicity detection kit (L-3224, Life Technologies). The method involves preparing a mixed solution of Calcein-AM and Ethidium homodimer-1 at concentrations of 0.5 μL / mL and 2 μL / mL, respectively. After washing the scaffold with PBS 7.4 buffer, the detection solution was added to the scaffold and incubated at 37°C for 20 minutes. Fluorescence images were detected using a microscope (Nikon Ti2-E inverted fluorescence microscope). Fluorescent staining of live cells was detected using an excitation wavelength of 488 nm, resulting in a green color. Dead cells were detected using an excitation wavelength of 633 nm, resulting in a red color. The number of live and dead cells was manually counted, and the cell viability was calculated by dividing the number of live cells by the total number of cells. The results were analyzed using ImageJ software.

[0136] The results are as follows Figure 6 As shown in Figure 2, (A) fluorescence microscopy images of live and dead cells in each group; (B) statistically analyzed cell survival rates after 7 days of in vitro culture. The results showed that the cell survival rate in each group reached approximately 85%, demonstrating the excellent biocompatibility of the hydrogel scaffold of the present invention.

[0137] In vitro chondrogenesis experiment of HAMA / Gel-lipo cells / hydrogel scaffolds

[0138] HAMA / Gel-lipo cells / hydrogel scaffolds were cultured at 37°C in a 5% CO2 environment. The culture medium was replaced every two days. On the 14th and 21st days, the cultured cells / hydrogel scaffolds were removed, fixed in 4% paraformaldehyde for one day, and then placed in a cryoembedding medium (OCT) for three days. Frozen sections were obtained by rapid freezing in liquid nitrogen and subjected to pathological morphological analysis (Alcian blue).

[0139] The results are as follows Figure 7 As shown, when cells were transplanted into the super-lubricating biohydrogel scaffold, the cells secreted more cartilage extraplasmic matrix in the high liposome concentration group.

[0140] Osteochondral defect repair experiment using HAMA / Gel-lipo cells / hydrogel scaffold

[0141] In order to verify the repair effect of the hydrogel scaffold in vivo when co-cultured with cells, the present invention constructed a mouse osteochondral defect model (refer to the prior art Wang, J., Zhang, F., Tsang, WP, Wan, C., & Wu, C. (2017). Fabrication of injectable high strength hydrogel based on 4-arm star PEG forcartilage tissue engineering. Biomaterials, 120, 11-21.). Pathological tissue sections proved that the model constructed a defect with a diameter of 1 mm and a depth of 1 mm at the distal femur of SCID mice, indicating that the model was successful. After the construction was completed, we took 1 μL of the HAMA / Gel-lipo / cell mixture in Example 5 and dripped it into and covered the defect, and then used a wavelength of 365 nm, 10 mW / cm 2 The cells were irradiated and cured under blue light for 5 minutes. After curing, they were sutured and the mice were observed and cultured for 4 and 8 weeks.

[0142] After the expiration, the osteochondral tissue of the sacrificed mice was removed and embedded in paraffin. The implantation site of the hydrogel scaffold was found through paraffin sectioning, and then pathological morphological analysis was performed. The cell morphology and adhesion to the surrounding tissue were observed and analyzed by hematoxylin-eosin staining, and the content of extracellular matrix (chitosan, glycosaminoglycan) secreted by the cells was detected by safranin-O staining. The corresponding antibodies (such as PRG4, Col II and MMP13) were selected to perform immunohistochemical analysis on the model to verify its repair effect on osteochondral defects.

[0143] The experimental steps of hematoxylin-eosin (H&E) staining are as follows:

[0144] Dewax the sections by sequentially placing them in xylene, anhydrous ethanol, 90% alcohol, 80% alcohol for 5 minutes, and 70% alcohol for 2 minutes each. Finally, rinse with tap water. Stain the dewaxed sections in hematoxylin solution for 3-5 minutes, then rinse with tap water. Observe the staining under a microscope. If the color is dark, rinse with tap water. If the color is lighter, continue restaining until the color is appropriate. Stain the sections in eosin solution for 0.5-1 minute. After completing both staining steps, place the sections in 70% alcohol, 80% alcohol, 90% alcohol, and xylene for 2 minutes each, then seal the sections.

[0145] The staining results in blue nuclei and red cytoplasm, which can help clearly show the morphological structure of cells and tissues.

[0146] The experimental steps of Safranin-O staining are as follows:

[0147] The sections were sequentially placed in xylene, anhydrous ethanol, 90% alcohol, 80% alcohol for 5 minutes, and 70% alcohol for 2 minutes each, and finally rinsed with tap water to complete the dewaxing process. The sections were then stained in Weigert's iron hematoxylin solution for 5 minutes. Differentiation was performed using acidic ethanol differentiation solution for 15 seconds; then, the sections were rinsed in distilled water for 1 minute; the sections were immersed in Fast Green stain for 1-2 minutes, and then rinsed in distilled water for 1 minute. The sections were immersed in Safranin-O stain for 5 minutes and then rinsed in distilled water for 1 minute. After staining, the sections were sequentially placed in 70% alcohol, 80% alcohol, 90% alcohol, and xylene for 2 minutes each, and finally, the sections were mounted. The staining results showed that the cartilage matrix appeared red, the chondrocyte nuclei were blue, the cytoplasm, muscle, collagen fibers, and bone tissue were gray-green, the cartilage cytoplasm was red, and the nuclei were gray-black.

[0148] The experimental steps of immunohistochemical staining (PRG4) are as follows:

[0149] Deparaffinize the sections by placing them in xylene, anhydrous ethanol, 90% alcohol, 80% alcohol for 5 minutes, and 70% alcohol for 2 minutes each. Finally, rinse with tap water to complete the dewaxing process. Antigen retrieval was performed using a heat-mediated method by placing the samples in a solution of 10mM sodium citrate, 0.05% Tween 20, pH 6.0, and maintaining the temperature at 98°C for 20 minutes. After removing the sample, use blocking solution (such as serum, protein, etc.) to block nonspecific binding sites to reduce background staining; incubate the specific first antibody Anti-Lubricin / MSF (ab28484, abcam) with the antigen on the tissue section to allow the antibody to fully bind to the antigen, generally at room temperature for 4 hours or 4°C overnight; use a second antibody with a marker (such as an enzyme) (IHC kit, ab269452, abcam) to bind to the first antibody to prepare for the color development step; through an enzyme substrate reaction (such as DAB), a visible color signal is formed on the tissue section to show the location and expression level of the antigen; use hematoxylin as a counterstain to better locate protein expression; finally, seal the slide and observe.

[0150] The results are as follows Figure 8 、 Figure 9 shown. Figure 8 In the figure, (A) Hematoxylin-eosin (H&E) staining confirmed that the experimental group adhered well to the native tissue and exhibited a significant repair effect; (B) Sudan red staining (Safranin-O) confirmed that the cells in the experimental group secreted more cartilage extracellular matrix, and overall chondrogenic differentiation was more obvious. The results showed that by constructing an osteochondral defect model in mice, a control group loaded with cells and an experimental group of super-lubricating hydrogel scaffolds were injected into the defect. After 8 weeks of observation, it was found that the experimental group had a significantly better repair effect at the defect than the control group.

[0151] Figure 9 To examine the effects of lubrication on cells in the experimental group, immunohistochemical staining of PRG4 was performed in an 8-week mouse osteochondral defect model. (A) PRG4 expression in the superficial layer of the experimental group was significantly higher than that in the defect and control groups, and approached the expression level of the SHAM group. (B) Negative control group without incubation with PRG4 antibody.

[0152] Comparative Example 1

[0153] Based on Example 5, the gelatin in Example 2 was replaced with an equal molar amount of double-bonded gelatin (GelMA), and the prepared double-bonded gelatin microspheres replaced an equal amount of gelatin microspheres. HSPC freeze-dried powder was added at a concentration of 50 mM to prepare the cell / hydrogel scaffold of Comparative Example 1.

[0154] Using it for the same performance test, the results show:

[0155] When the gelatin microspheres were replaced with an equimolar amount of GelMA microspheres, the hydrogel scaffold was physically characterized and tested. The results showed that the overall Young's modulus of the hydrogel was slightly improved (the structure of the scaffold microenvironment was denser), but the COF of the scaffold did not change significantly compared with the hydrogel scaffold in the present invention.

[0156] After three-dimensional in vitro culture, the results showed that the bioscaffold with GelMA microspheres showed strong biocompatibility, and the stem cells showed obvious proliferation in the scaffold. However, subsequent observations and tests found that after replacing gelatin with GelMA, the cells did not show obvious aggregation or suspended growth, and were more inclined to grow adherently. Through further observation and analysis, it was found that in the bioscaffold with GelMA microspheres, compared with the hydrogel of the present invention, the stem cells did not show the unique morphology of chondrocytes (elliptical), but instead showed the characteristics of osteoblasts (flat polygonal shape); through staining of the extracellular matrix, it was also found that the stem cells attached to GelMA secreted less cartilage extracellular matrix (type II collagen, proteoglycans...) during growth, but the extracellular matrix of osteoblasts (type I collagen, osteonectin) increased significantly.

[0157] The above results show that when gelatin microspheres are replaced with GelMA, the Young's modulus of the overall bioscaffold will be slightly improved due to the denser internal structure, but the COF does not change significantly compared with the hydrogel scaffold of the present invention; there is no significant difference in the growth and proliferation of stem cells, but there is a significant effect on the differentiation of stem cells. In the presence of the hydrogel of the present invention, stem cells will significantly differentiate towards the cartilage phase, but when gelatin is replaced with GelMA, the cells are more inclined to differentiate towards the osteogenic phase.

[0158] Comparative Example 2

[0159] The macro-micro composite hydrogel scaffold is prepared by referring to the method of the prior art CN18178734B, comprising:

[0160] S1. Preparation of GelMA micro-hydrogel scaffolds

[0161] (1) Prepare a 0.5 g / mL GelMA solution in deionized water, add 3 wt% phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt, and transfer the solution into one of the syringes of a microinjection pump, which is recorded as the aqueous phase.

[0162] 40 mL of olive oil (containing 35% by volume of Span 80) was transferred into another syringe of the microinjection pump, which was recorded as the oil phase.

[0163] (2) The aqueous phase and the oil phase were injected at a rate of 0.4 mL / h and 120 mL / h, respectively, so that they converged in the T-shaped channel of the injection needle, generating shear force to produce GelMA microgels.

[0164] The microgel was collected in a small glass bottle containing the oil phase and cured under UV light at 365 nm, 180 mW / cm' for 30 s. After standing, the upper oil phase was removed by aspiration and the microgel was washed with acetone several times to obtain a microscopic hydrogel, i.e., the microgel. The microgel was freeze-dried and set aside.

[0165] S2, Rhodamine B red fluorescence labeled microgel GelMA

[0166] (1) 0.15 g of rhodamine B was dissolved in 10 mL of N,N-dimethylformamide, followed by the addition of 0.2 g of N-hydroxysuccinimide and 0.2 g of 1-ethyl-3-dimethylaminopropyl-carbodiimide hydrochloride, stirring to dissolve, and reacting at room temperature for 4 h. This was recorded as the activation solution.

[0167] (2) Dissolve 1 g of GelMA microgel in 20 mL of deionized water, heat in a water bath, and stir magnetically. Add 3 mL of activation solution and allow to react in the dark for 24 h. Terminate the reaction by adding 80 mL of deionized water. Place the solution in a dialysis bag and dialyze against deionized water (in the dark). Centrifuge the dialyzed solution to remove the precipitate, transfer the supernatant to a culture dish, and freeze-dry to obtain rhodamine-labeled microgel.

[0168] S3. Construction of macro-micro composite hydrogel scaffolds

[0169] Fifty freeze-dried GelMA microgels were added to an aqueous solution with a total mass concentration of 6 wt% of HAMA and GelMA, and 2 wt% of LAP blue light initiator was added. The mixture was shaken and mixed to obtain a composite solution. The composite solution was dropped into a mold and gently stirred with an injection needle to mix the GelMA microgels evenly in the gel-making solution. The solution was then irradiated and cured under 405 nm, 10 mW / cm* blue light for 30 s to form a macro-micro composite hydrogel scaffold.

[0170] Using it for the same performance test, the results show:

[0171] Physical characterization tests were performed on the hydrogel, and we found that the COF of the hydrogel scaffold (μ is 0.08-0.2) is significantly higher than that of the hydrogel scaffold of the present invention, and much higher than that of natural cartilage (μ value is 0.002-0.03).

[0172] After in vitro three-dimensional culture, the results showed that the hydrogel bioscaffold of comparative example 2 showed strong biocompatibility, and the stem cells maintained a high survival rate in the scaffold, and obvious proliferation was observed. However, when the number of days of culture was extended to 14 and 21 days, the cells did not show obvious aggregation or suspension growth trends, but tended to grow attached to the wall; after further pathological detection and analysis of the bioscaffold, the results showed that the cells in the bioscaffold did not show the unique morphology of chondrocytes (elliptical), but instead showed the characteristics of osteoblasts (flat polygonal shape); by staining the extracellular matrix, it was also found that the stem cells in the hydrogel of comparative example 2 secreted less cartilage extracellular matrix (type II collagen, proteoglycans...) during growth. Specific immunohistochemical staining was performed on PRG4, which is related to cartilage surface lubrication. The results showed that, compared with the present invention, PRG4 showed a significant downward trend, but the extracellular matrix of osteoblasts (type I collagen, osteonectin) increased significantly.

[0173] The results showed that the hydrogel in Comparative Example 2 significantly promoted the differentiation of stem cells toward the osteogenic phase and secreted more extracellular matrix related to osteogenesis. However, it failed to promote the differentiation of stem cells toward the chondrogenic phase as the hydrogel of the present invention, and failed to induce cells to express more chondrocyte-related extracellular matrix. Therefore, the hydrogel in Comparative Example 2 is not suitable for articular cartilage repair.

[0174] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of a three-dimensional biohydrogel in the preparation of a cartilage repair preparation, characterized in that: The three-dimensional biohydrogel comprises, by weight, 1-5 parts of methacrylic anhydride hyaluronic acid HAMA, 0.05-0.15 parts of an initiator, 3-8 parts of hydrogenated soybean phosphatidylcholine liposomes, and 7-13 parts of a stem cell solution. The preparation method of the stem cell solution comprises the following steps: mixing gelatin and water to prepare a gelatin solution; stirring the mixture at 40-60° C. to prepare a dispersed phase; adding an emulsifier to the oil to prepare a continuous phase; preparing the dispersed phase and the continuous phase into uniform droplets using microfluidics technology; freeze-drying the droplets to obtain gelatin microspheres; and adding 5*10 5 cells / mL of stem cells, incubated at 25-37°C for 2-4 hours to obtain a stem cell solution; In the process of preparing uniform droplets using microfluidic technology, the flow rate of the dispersed phase is 30-40 μL / min; the flow rate of the continuous phase is 500-600 μL / min; The particle size of gelatin microspheres is 40-60 μm.

2. The use according to claim 1, characterized in that The mass concentration of gelatin in the gelatin solution is 5%-10%.

3. The use according to claim 1, characterized in that The method for preparing the three-dimensional biohydrogel comprises the following steps: S1. adding an initiator to methacrylic anhydride-modified hyaluronic acid (HAMA) to obtain a hydrogel precursor solution; S2. Add hydrogenated soybean phosphatidylcholine liposomes and stem cell solution to the hydrogel precursor solution, mix them evenly, and obtain a composite solution; transfer the solution to a mold and form a three-dimensional biohydrogel by photocuring.

4. The use according to claim 3, characterized in that The hydrogenated soybean phosphatidylcholine liposomes are first dispersed and then added dropwise into the hydrogel precursor solution.

5. The use according to claim 3, characterized in that Light curing is performed under 365nm, 8mW / cm²-12mW / cm² blue light for 4-8 minutes.

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