Injectable double-network hydrogel stent as well as preparation method and application thereof

Abstract

The invention provides an injectable double-network hydrogel stent as well as a preparation method and application thereof, and belongs to the technical field of preparation of hydrogel implant materials. The preparation method of the injectable double-network hydrogel scaffold comprises the following steps: (1) dissolving bacterial cellulose, and adding an oxidizing agent NaIO4 to carry out oxidation reaction to prepare oxidized bacterial cellulose; (2) preparing a mixed solution from the methacrylic anhydride gelatin hydrogel solution, oxidized bacterial cellulose and sodium alginate, and performing photo-crosslinking curing to prepare double-network hydrogel; and (3) dissolving the dual-network hydrogel obtained in the step (2) in water, and then mixing with a growth factor IGF-1 to prepare the IGF-1 loaded dual-network hydrogel stent. The micro-environment can be adjusted, the mechanical strength of the micro-environment can be enhanced, the internal soft matrix material can promote cartilage regeneration of the growth plate, the external gradually-reinforced material can block an injured part and create a hypoxic micro-environment, and growth cartilage repair is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel implant materials, and in particular relates to an injectable double-network hydrogel scaffold and a preparation method and application thereof. Background Art

[0002] 30% of pediatric bone trauma will involve the growth plate, and growth plate injury is the most common cause of secondary skeletal deformities in children, and there is currently a lack of effective treatment. After a child's fracture, the growth plate is damaged and the cartilage cannot be regenerated. At the same time, new bone tissue replaces the cartilage to form a bone bridge. Similarly, cumulative infection, tumors or iatrogenic damage to the growth plate will lead to the appearance of a bone bridge. When the bone bridge occupies less than 50% of the area of ​​the growth plate, surgical removal of the bone bridge is required to insert different insertion materials, including fat, bone wax, muscle or polymer silicone materials to prevent bone bridge regeneration.

[0003] However, the clinical success rate of surgical removal of bone bridges is less than 35%, because the currently available implant materials do not integrate well with the host tissue, often leading to subsequent complications. When the bone bridge occupies more than 50% of the growth plate, corrective surgery and limb lengthening surgery are required clinically. Again, the results are not satisfactory. Unfortunately, clinical treatment can also lead to secondary injury or recurrence of bone bridges, and serious complications such as unequal length of lower limbs, premature epiphysis closure, and valgus and valgus deformity of limbs may still occur in the later stage. At this time, only more traumatic osteotomy can be performed to correct it, but it may also bring serious complications such as growth disorders, limited joint movement, and limb deformities. It causes serious psychological and physiological damage to children, endangers family health and harmony, and endangers the future development of the motherland.

[0004] Therefore, there is an urgent need to develop a treatment method or implant for growth plate damage that can prevent the formation of bone bridges and regenerate healthy epiphyseal tissue, thereby restoring normal bone growth. However, research in this area still needs to be continued.

[0005] Endochondral bone formation is an important process in vertebrate bone formation and is highly dependent on the correct function of growth plate chondrocytes. Growth plate cartilage can be divided into four zones: resting zone, proliferating zone, hypertrophic zone, and calcification zone. The chondrocytes in the resting zone closest to the epiphysis are hyaline chondrocytes, which are round in shape and are considered to be the progenitor cell group of the growth plate; the cells in the proliferating zone are flatter, and the cells in this area are in a rapid proliferation phase and produce proteoglycans and type II collagen, which play a key role in the longitudinal growth of bones; the hypertrophic zone contains chondrocytes, which stop proliferating and begin to grow rapidly, expand in size, and produce glycogen to increase the volume of extracellular matrix. The proliferation of these cells determines the longitudinal growth of the bone, and the deposited matrix provides a scaffold for future bone formation. However, these two energy-dependent assimilation processes are carried out in an avascular environment. During embryonic development, chondrocytes in the center of long bones are avascular and therefore in a hypoxic stage, at which time hypoxia-inducible factor (HIF-1) appears and is responsible for coordinating the response among chondrocytes. Under hypoxic conditions, HIF-1 is stabilized and able to translocate to the nucleus, heterodimerize with the β subunit, and initiate its transcriptional program together with other auxiliary factors. Therefore, the hypoxic microenvironment is essential for the survival of growth plate chondrocytes.

[0006] Studies have shown that when the growth plate is injured, oxygen-rich hematoma accumulates at the damaged site, causing the microenvironment for growth plate cartilage regeneration to be destroyed, while endothelial cells and osteoblasts adapted to the oxygen-rich microenvironment will continue to proliferate and differentiate into osteoblasts, leading to the formation of bone bridges. Other evidence shows that BMSCs flow into the injured site from the surrounding area, thereby increasing the expression of osteogenic markers and reducing the expression of chondrogenic markers. The development of the growth plate is a complex multi-level regulatory process, among which IGF-1 is an important growth factor that has been widely studied and has a clear role in promoting the regeneration of the growth plate. Previous studies have confirmed that IGF-1 can not only stimulate chondrocytes to synthesize matrix proteins such as Col II and proteoglycans, but also inhibit the degradation and apoptosis of chondrocytes during cartilage injury by blocking the effects of IL-1 or TNF-α. In a clinical trial, IGF-1 was used to treat short children for 1 year, and no adverse events were reported, suggesting that IGF-1 may have clinical application potential.

[0007] In order to inhibit the formation of bone bridges, researchers have tried a variety of materials. For example, Michael et al. used sodium alginate and chitosan to make an injectable hydrogel, and proved in vitro and in vivo that this hydrogel can promote the regeneration of growth plate cartilage. A study inhibited the formation of bone bridges after sheep growth plate injury by using a gelatin sponge scaffold combined with TGF-β1 and autologous bone marrow mesenchymal stem cells. Chen et al. embedded the collected periosteum-derived MSCs in agarose and further transplanted them into the growth plate defect, effectively correcting the angular deformity and growth arrest. Tomaszewski et al. removed the medial side of the proximal tibial growth plate of rabbits, cultured chondrocytes, and re-implanted the cultured chondrocytes into the growth plate defect area. Both histological and radiological results showed that implanting autologous chondrocytes can prevent the formation of bone bridges and avoid the occurrence of growth arrest. Li et al. transplanted alginate semipermeable membrane loaded with allogeneic chondrocytes obtained from the distal femoral growth plate into a growth plate injury model. After 16 weeks, the length difference and angular deformity of the experimental group were less than those of the other groups. Histological results also showed that the newly formed chondrocytes at the injury site formed a columnar arrangement.

[0008] Growth plate damage will lead to inflammatory response, which will disrupt the balance of ECM synthesis and decomposition. Inflammatory factors will stimulate damaged chondrocytes to produce MMPs, while inhibiting the expression of SOX-9, further exacerbating the decomposition of chondrocyte ECM. Guan et al. synthesized chondroitin sulfate-methacrylic anhydride gelatin hydrogel, carried exosomes and implanted them into the damaged area of ​​the growth plate to inhibit the inflammatory response and promote cartilage regeneration.

[0009] However, the various implant materials prepared by the above-mentioned studies all showed poor effects in promoting cartilage differentiation and could not be used well for the recovery of growth plate injuries. For example, the combination of some hydrogel materials and growth factors has poor mechanical properties and cannot meet the mechanical strength requirements of cartilage implants; some hydrogel materials, although they have good mechanical strength, cannot form a large amount of cartilage in the body, but show the ability of osteogenic differentiation; some hydrogel materials cannot adapt to the avascular and hypoxic characteristics of cartilage tissue and cannot form a hypoxic environment well, resulting in poor healing effect of implant materials on growth plate cartilage.

[0010] Therefore, it is urgent to develop an implant material that can not only meet the mechanical strength requirements of cartilage implants, but also adapt well to the hypoxic environment of cartilage tissue, and promote chondrogenic differentiation rather than osteogenic differentiation, so as to achieve efficient treatment of growth plate cartilage injuries in children. Summary of the invention

[0011] The present invention is to solve the above technical problems, thereby providing an injectable double-network hydrogel scaffold and its preparation method and application. The technical purpose of the present invention is to provide a hydrogel implant material to solve the problems that the existing hydrogel implant materials have insufficient mechanical strength on the one hand and cannot meet the strength requirements of bone implant materials, and on the other hand, they cannot form an oxygen-deficient environment that meets the growth of cartilage tissue, so they are not effective in treating cartilage damage. The third aspect is that they cannot promote cartilage differentiation well but tend to promote osteogenic differentiation, resulting in the inability to form a large amount of growth plate cartilage in the body to achieve the purpose of growth plate cartilage repair.

[0012] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0013] The present invention first provides a method for preparing an injectable double-network hydrogel scaffold, which comprises the following steps:

[0014] (1) Dissolve bacterial cellulose and add oxidant NaIO 4 Performing an oxidation reaction to prepare oxidized bacterial cellulose;

[0015] (2) preparing a mixed solution of methacrylic anhydride gelatin hydrogel solution, oxidized bacterial cellulose and sodium alginate, and performing photo-crosslinking and curing to prepare a double network hydrogel;

[0016] (3) Dissolving the double network hydrogel obtained in step (2) in water, and then mixing it with the growth factor IGF-1 to prepare a double network hydrogel scaffold loaded with IGF-1.

[0017] The present invention prepares a hydrogel that can adjust the microenvironment and enhance its own strength. By using the aldehyde group (-CHO) of aldehyde-modified bacterial cellulose (DBNC) and the amino group (-NH 2 ) to form Schiff base bonds, building the first layer of network structure. At the same time, according to the characteristics of sodium alginate (Alg) and the charged properties of the material, Alg can autonomously recruit Ca in the bone tissue that contacts the upper and lower surfaces of the material in the body. 2+ They combine to form an "egg-box" structure, and at the same time attract the negative charge of DBNC to form an interpenetrating structure with the first layer of network, further strengthening the hydrogel to form a "hamburger"-like structure, in which the soft matrix material inside can promote the regeneration of growth plate cartilage, while the gradually strengthened material on the outside can play an important role in sealing the damaged area and creating an oxygen-deficient microenvironment.

[0018] In vitro experiments have shown that this hydrogel scaffold can release IGF-1 growth factor to further activate the PI3K-Akt pathway to promote subsequent cartilage differentiation and regeneration. 2+In the presence of , this hydrogel can still reduce the expression of related osteogenic genes (RUNX2, BMP2 and OST). In in vivo experiments, this hydrogel scaffold can create a hypoxic microenvironment after implantation and specifically express growth plate related genes (Col10a1, KLF13, BMP2 and RUNX3) to promote growth plate cartilage regeneration.

[0019] The preparation method provided by the present invention can form a double-layer hydrogel network scaffold with a dense exterior and soft interior by allowing the raw materials to recruit calcium ions in the body after being injected into the body. The hydrogel scaffold can well meet the mechanical property requirements of bone implant materials.

[0020] The inventor initially prepared hydrogel by using hyaluronic acid as the base material and loading growth factors, but found that the mechanical strength of the hydrogel material was too low to meet the strength requirements of bone implants. In order to improve the mechanical properties of the hydrogel material, the inventor tried to mix calcium ions with the hydrogel base material, and found that the mechanical properties of the hydrogel material prepared in this way were still poor, and the density formed on the surface of the material was large. Even if the growth factor was loaded, the internal material could not promote cartilage differentiation.

[0021] In order to further improve the effect of the material in promoting cartilage differentiation, the inventors adopted the method of improving the carrier and adding active substances. However, when trying to use the method of directly mixing and cross-linking the sodium alginate solution with calcium ions in vitro, although a dense barrier layer can be formed, the material inside still tends to differentiate into osteoblasts and cannot form cartilage differentiation in large quantities, resulting in poor repair effect on growth plate cartilage damage.

[0022] Finally, through a lot of exploration, the inventors obtained the above-mentioned preparation method. Instead of adding calcium ions to the GelMA hydrogel solution, they only chose to add sodium alginate and oxidized bacterial cellulose. It was found that the hydrogel material can continuously recruit calcium ions in the body to form a dense barrier layer on the surface, and the growth factors loaded inside the material can still remain soft, thereby promoting cartilage differentiation and enhancing the therapeutic effect of drugs on growth plate cartilage damage.

[0023] Furthermore, in step (1), the bacterial cellulose and NaIO 4 The weight ratio is 2:1.

[0024] Furthermore, the temperature of the oxidation reaction in step (1) is 40° C., the reaction time is 14 h, and the reaction pH is 6.0.

[0025] Furthermore, the method for preparing the methacrylic anhydride gelatin hydrogel in step (2) is to disperse gelatin in a carbonate buffer solution, then react with methyl acrylate, and prepare the methacrylic anhydride gelatin hydrogel by centrifugal dialysis.

[0026] Furthermore, in step (2), the weight ratio of the methacrylic anhydride gelatin hydrogel solution, oxidized bacterial cellulose and sodium alginate is 7.5:1:1.2.

[0027] Furthermore, the amount of IGF-1 added in step (3) is 100 ng / mL.

[0028] The second object of the present invention is to provide an injectable double-network hydrogel scaffold prepared by any of the above methods.

[0029] Furthermore, the double-network hydrogel scaffold has the ability to autonomously recruit calcium ions in the body, thereby achieving self-enhancement of the hydrogel's mechanical properties. The material matrix of the part close to the bone gradually strengthens, while the part close to the growth plate cartilage maintains the strength of the soft matrix, forming a "hamburger"-like structure, which can match the pathological state of the growth plate after injury, thereby promoting the regeneration of the growth plate cartilage.

[0030] The third object of the present invention is to provide the use of the above-mentioned injectable double-network hydrogel scaffold in the preparation of implant materials for treating growth plate cartilage injuries.

[0031] A fourth object of the present invention is to provide an application of the above-mentioned injectable double-network hydrogel scaffold in the preparation of a drug for treating growth plate cartilage damage.

[0032] The beneficial effects of the present invention are as follows:

[0033] (1) The present invention prepares an injectable double-network hydrogel scaffold material that can be directly injected into the cartilage damage site to effectively repair the growth plate cartilage damage;

[0034] (2) The hydrogel material prepared by the present invention can recruit calcium ions in situ in vivo, thereby forming a double network structure with a dense surface and a soft interior, and has little effect on the growth factors loaded inside the material, and will not affect the effect of the growth factors in promoting cartilage differentiation;

[0035] (3) The hydrogel material of the present invention has excellent mechanical strength and can meet the requirements of bone implant materials for high mechanical properties;

[0036] (4) The hydrogel material of the present invention can block the damaged area and create an oxygen-deficient microenvironment, and efficiently promote cartilage differentiation, thereby greatly improving the therapeutic effect of existing hydrogel materials on growth plate cartilage damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the present invention using GelMA, sodium alginate and oxidized bacterial cellulose to prepare a double network hydrogel, and locally injecting the hydrogel to block the damaged part of the growth plate to create an oxygen-deficient microenvironment and promote the regeneration and proliferation of growth plate chondrocytes.

[0038] Figure 2 A: stress-strain curve of hydrogel composed of materials with different concentrations; B: Young's modulus of hydrogel composed of materials with different concentrations; C: stress-strain diagram of hydrogel composed of materials with different concentrations.

[0039] Figure 3 A: The microstructure of calcium-containing self-reinforced and calcium-free hydrogel materials under electron microscope and the morphological observation of cell growth on hydrogels of different material groups; B: The swelling curve of the hydrogel; C: FTIR graph.

[0040] Figure 4 A: Live and dead cell staining results after co-culture of cells with hydrogels of different materials for 24 and 48 hours; B: CCK8 experimental results of different groups of materials in the presence and absence of calcium.

[0041] Figure 5 A: Schematic diagram of RNA-seq cultured in hydrogels of different materials; B: Veen diagram; C: Volcano diagram of differentially expressed genes between blank group and GDAI; D: Schematic diagram of RNA-seq cultured in hydrogels of different materials; 2+ Volcano plot of differentially expressed genes; E: GO and KEGG analysis of differentially expressed genes between the GDAI group and the blank group (F).

[0042] Figure 6 GDAI+Ca 2+ GO (A) and KEGG analysis of differentially expressed genes between the treated group and the blank group (B); C: Expression of osteogenic genes in different material groups in the presence of calcium ions.

[0043] Figure 7 A: Schematic diagram of the in vivo experiment; B: MRI cross-sectional, coronal and sagittal planes to observe the bone bridge formation after growth plate injury; C: ImageJ was used to perform statistics on the MRI results.

[0044] Figure 8 A: HE and TB staining of each group; B: Quantitative analysis of HE and TB results using ImageJ.

[0045] Fig. 9 A: The expression of Sox-9 among the groups; B: The expression of Col II among the groups; C: The expression of Hif-1α among the groups.

[0046] Fig.10A: Schematic diagram of RNA-seq after animal experiments; B: Volcano plot and GO analysis of differentially expressed genes between the GDAI group and the blank group (C).

[0047] Fig.11 A: KEGG analysis of differentially expressed genes between the GDAI group and the blank group; B: STRING analysis of differentially expressed genes between the GDAI group and the blank group.

[0048] Fig.12 A: Each partition of the growth plate; B: The representative genes that have been reported in each partition; C: RT-PCR verification of the expression of key genes in the growth plate.

[0049] Fig.13 Macroscopic image of the GD hydrogel scaffold; Schiff bonds (DBNC-CH=N-GelMA) are formed between the amino groups (GelMA hydrogel) and the aldehyde groups (DBNC).

[0050] Fig.14 With or without Ca 2+ Characterization of hydrogels; A: GDAI+Ca 2+ hydrogel; B: GDAI hydrogel.

[0051] Fig.15 The presence or absence of Ca observed by SEM 2+ Microscopic morphology of the hydrogel scaffold.

[0052] Fig.16 IGF-1 in GDAI and GDAI+Ca 2+ Release profiles from hydrogels.

[0053] Fig.17 GDAI+Ca 2+ and without Ca 2+ String protein network interaction analysis. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0055] Example 1

[0056] 1. Materials and Methods

[0057] 1. Preparation of Hydrogel

[0058] 1.1 Synthesis of GelMA

[0059] Weigh 20 g of gelatin and disperse it in 200 mL of carbonate (pH 9.0) buffer solution, place it in a 50°C oil bath and heat and stir until it is completely dissolved to prepare a 10 wt% gel solution;

[0060] Carbonate buffer: NaCO 3 (0.3427 g) and NaHCO 3 (3.0915g) was dissolved in 200mL of deionized water. 2mL of MA (methyl acrylate) was extracted with a syringe, and MA was slowly added to Gel at a speed of 0.2ml / min using a microinjection pump, paying attention to avoiding light; after adding MA, the reaction was continued in an oil bath for 3 hours, and then 100mL of PBS was added to terminate the reaction; after the reaction was terminated, the unreacted MA was removed by centrifugation at 7000rpm for 15min to obtain GelMA (methacrylic anhydride gelatin), and then GelMA was divided into dialysis bags (MWCO 3500), dialyzed at 38℃ for 2 days, and freeze-dried to obtain the product.

[0061] 1.2 Synthesis of DBNC (aldehyde-modified bacterial cellulose)

[0062] Weigh 1g of BNC (bacterial cellulose) dry film, cut it into pieces, place it in 200mL deionized water for homogenization, and transfer the slurry into a 500mL round-bottom flask. Wrap the round-bottom flask with tin foil to avoid light, and add the oxidant NaIO 4 (W NaIO4 / W BNC The reaction was carried out at a temperature of 40°C and a pH of 6.0 for 14 h. After the reaction was completed, the precipitate was collected by suction filtration and added with 0.1 mol / L ethylene glycol solution to continue the reaction for more than 30 min to remove the unreacted NaIO 4 After the reaction, the mixture was filtered and washed with deionized water for 5 times. The residue was collected and freeze-dried to obtain DBNC.

[0063] 1.3 Assembly of hydrogel

[0064] Take a certain amount of GelMA, dissolve it in ultrapure water, and make a hydrogel solution with a concentration of 5-10wt%. Take a certain amount of DBNC, dissolve it in the above hydrogel solution, and make a mixed hydrogel with a concentration of 0.5-1wt% (referred to as GD hydrogel). Take a certain amount of Alg (sodium alginate), dissolve it in the above mixed hydrogel to make its concentration 1.2wt%. After mixing various materials with an ultrasonic cell disruption instrument, cross-link them with ultraviolet light to prepare a double network hydrogel (referred to as GDA hydrogel). After optimizing the concentration of DBNC and GelMA, 100ng / mL of IGF-1, 1.2wt% of Alg and a certain amount of DBNC and GelMA are mixed, and then cross-linked with ultraviolet light to prepare a double network hydrogel loaded with IGF-1, referred to as GDAI hydrogel.

[0065] 2. Characterization of Hydrogels

[0066] 2.1 Determination of material concentration

[0067] The prepared hydrogels were divided into 9 groups, and the mechanical properties were tested, and the elongation at break curves were drawn. The Young's modulus was then calculated based on the results. The differences in mechanical properties between the groups were analyzed to select the appropriate concentrations of various materials.

[0068] 2.2 Characterization of different groups of hydrogels

[0069] SEM was used to observe the microstructure of the hydrogel and the cell growth gap; ATR-FTIR recorded the chemical structure of different groups of hydrogels after freeze-drying. The swelling rate of different hydrogels was measured by soaking the freeze-dried materials in ultrapure water at 37°C and shaking at 80rpm. The mass was measured after absorbing the surface moisture after 1h, 2h, 4h, 8h, 16h, 24h, 48h, 4 days, and 7 days. The release of IGF-1 (growth factor) in various materials at different time points was determined by ELISA to determine the encapsulation rate and release performance of the drug.

[0070] 3. Investigation of hydrogel properties in vitro

[0071] 3.1 Biocompatibility and effects on cell morphology

[0072] The sterilized materials were placed in a 6-well plate and grouped into GD, GDA, GDAI without calcium and GD, GDA, GDAI with calcium. Three parallel controls were set for each material, and UV sterilization was performed for more than 2 hours after cross-linking. Among them, GD represents GelMA hydrogel loaded with DBNC; GDA represents GelMA hydrogel loaded with DBNC and Alg; GDAI represents GelMA hydrogel loaded with DBNC, Alg and IGF-1.

[0073] The third generation rat BMSCs were cultured at 1×10 5The samples were inoculated at a density of 1 / mL in α-MEM containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% CO 2 Incubate for 1, 3 and 5 days. After co-culture, wash with PBS 3 times, co-incubate with a mixture of 40μL CCK-8 and 360μL α-MEM for 1h, and then transfer 100μL of the suspension to a 96-well plate, and measure the absorbance at 450nm to evaluate the cytotoxicity of the material. The results are expressed as mean ± standard deviation (SD). Live / dead cell staining was performed after 24h and 48h of co-culture using Transwell culture dishes to further evaluate the biocompatibility of the material. In order to study the morphology and state of single cells after co-culture, the cells were fixed after 7d of co-culture, and the changes in the materials and cells were further observed using SEM.

[0074] 3.2 Transcriptome analysis

[0075] After 7 days of co-culture of materials and cells, transcriptome analysis was performed. 2+ The tissue samples of the group were ground under liquid nitrogen. Total RNA was isolated using RNAmini kit (Qiagen, Germany). RNA quality was checked by gel electrophoresis and Qubit (Thermo, Waltham, MA, USA). RNA-seq libraries were constructed using TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA) and sequenced using Illumina Novasek 6000 instrument. Transcriptome sequencing was performed by Sevier Biotechnology Co., Ltd. (Wuhan, China). The expression of transcripts was calculated using Perl's FPKM (fragments per kilobyte of exon models per million mapped reads). The differentially expressed transcripts (DETs) between different time points (12hpt vs. 0hpt, 36hpt vs. 0hpt, 72hpt vs. 0hpt) were determined using the MA-plot-based random sampling method (MARS) in the DEGseq software package. DEGs, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis were performed using R software. p value < 0.05 and FoldChange > 2 were set as the thresholds for significant differential expression or differential enrichment.

[0076] PPI analysis of differentially expressed genes was based on the STRING database, which contains known and predicted protein-protein interactions, and networks were built based on known interactions of selected reference species.

[0077] 3.3RT-qPCR

[0078] To verify whether the hydrogels in different groups can inhibit osteogenesis in the presence of calcium ions, we performed RT-qPCR. cDNA was synthesized using the Servicebio for qPCR kit (Saiweier, China) and RT-qPCR was performed using a Bio-rad PCR instrument. The results were based on GAPDH as the standard and 2 -△△Ct Method for analysis.

[0079] 4. Investigation of hydrogel performance in vivo

[0080] 4.1 Rat animal model of growth plate injury

[0081] Sixteen rats of about 5 weeks old were prepared and divided equally into a blank group, a GD group, a GDA group, and a GDAI group.

[0082] A rat proximal tibial growth plate injury model was prepared. The rats were anesthetized with isoflurane, and the animals were moved to the surgical site and laid flat on a warm pad and an absorbent pad. The hair of the entire hind leg from the medial malleolus to the pelvis was shaved with an electric shaver. The surgical site was cleaned by first wiping the entire leg, abdomen, and genitals with an alcohol swab, and then wiping with gauze soaked in povidone iodine. A drape was laid, and a 2 cm incision was made distally starting from the lower edge of the patella. The tibial fascia and soft tissue were gently dissected or scraped with a scalpel. The 1.6 mm Kirschner wire was placed perpendicular to the tibial shaft and drilled slowly. Be careful not to insert it too deep, generally about 1 cm deep. Gauze was applied with pressure to stop bleeding, followed by injection and cross-linking of the hydrogel, and finally the wound was sutured.

[0083] 4.2 Animal MRI examination and specific staining

[0084] In vivo MRI was used to assess growth plate damage and regeneration at 8 weeks with the following settings:

[0085] T1-weighted: SE (TE / TR=10 / 300, FOV=40x20mm, Matrix=256×128, NEX=10, Res.156um, Acq.Time 6:42min);

[0086] T2-weighted: SE (TE / TR=73.8 / 3100, FOV=40x20mm, Matrix=256×128, NEX=20, ETL=16, Res.156um, Acq.Time 10:00min).

[0087] Rats were killed after 8 weeks, and proximal tibia specimens were collected for immunohistochemical staining. The collected specimens were fixed and decalcified with 10% formic acid at room temperature for 2 weeks, then dehydrated with ethanol gradient and embedded in paraffin blocks. The embedded specimens were cut into 5 μm thick histological sections along the center of the defect area, and the damage and regeneration of growth plate cartilage were evaluated by hematoxylin and eosin (H&E) and toluidine blue (TB) staining. At the same time, Sox-9 immunohistochemical staining was used to evaluate the expression of chondrogenic genes, Col II was used to evaluate the degree of endochondral ossification, and HiF1α was used to evaluate the local hypoxic microenvironment.

[0088] 4.3 Transcriptome analysis

[0089] The growth plate tissues of the rats in the blank group and the GDAI group were taken for RNA-seq detection. The specific steps were the same as above.

[0090] 4.4 RT-qPCR

[0091] To verify the results of RNA-seq, RT-qPCR assay was performed.

[0092] 5. Statistics and Analysis

[0093] Graphpadprism (v9.0 CA) was used to perform statistical analysis of the experimental results. All data are expressed as mean ± standard deviation. Multiple experimental groups were analyzed using one-way analysis of variance (ANOVA). R software was used to perform statistical analysis of RNA-seq results. The statistical tests performed in this study are shown in the figure: *0.01 <p<0.05;**0.001<p<0.01;***0.0001<p<0.001;****p<0.0001。

[0094] 2. Experimental Results

[0095] 1. Concentration selection of double network hydrogel

[0096] In this experiment, different materials were mixed in different proportions to obtain different hydrogels, which were compressed repeatedly for 10 cycles, and the corresponding stress-strain diagrams were produced based on the results ( Figure 2 According to the results, when the concentration of other materials remains unchanged and the concentration of GelMA gradually increases, the stress of the hydrogel gradually increases during the deformation of 50%, indicating that the strength of the hydrogel gradually increases; when the concentration of GelMA remains unchanged and the concentration of DBNC gradually increases, the stress of the hydrogel does not change much when subjected to the same strain.

[0097] Next, the Young's modulus and maximum stress-strain results of the above hydrogels with different concentrations were calculated ( Figure 2B and C). It indicates that although the hydrogel has a higher strength when the concentration of GelMA is maintained at 10%, its elasticity is poor, with an elastic deformation of only about 50%. When the concentration of GelMA is 7.5%, the hydrogel can have a good elastic deformation effect while maintaining mechanical strength, reaching about 85%. The increase in the concentration of DBNC can increase its overall Young's modulus and mechanical properties. Therefore, according to the above experimental results, the concentration of the hydrogel material is 7.5% GelMA + 1% DBNC + 1.2% Alg (the above concentrations are all measured in mass volume percentage).

[0098] 2. Hydrogel Characterization

[0099] 2.1 Morphology

[0100] The present invention forms a Schiff base bond (DBNC-CH=N-GelMA) by using the amino group in GelMA hydrogel and the aldehyde group in DBNC. Fig.13 As shown in the figure, the first layer of network structure is formed. On this basis, Alg material is further added to recruit free calcium ions in the bones at the upper and lower ends of the damaged growth plate channel to undergo rapid cross-linking, forming a dense network structure at both ends, and the middle material is a loose macroporous structure, forming a "hamburger"-like structure (adding Ca 2+ and without Ca addition 2+ The macroscopic picture is further shown as Fig.14 shown).

[0101] SEM observation can further clarify the internal structure of the hydrogel. Fig.15 It shows that when there is no calcium ion, after adding DBNC to GelMA hydrogel, its internal fiber structure is obvious, and when Alg is added, the microscopic morphology does not change much; when calcium ions are present, obvious cross-linking can be seen on the surface of the hydrogel, which appears smoother and has denser internal connections. Figure 3 As shown in A, the internal structure of the part in contact with calcium ions is densely connected, which is in sharp contrast to the macroporous structure inside it. This further proves that the hydrogel constructed by the present invention can form a dense structure on the surface in contact with calcium ions, while the internal structure remains loose and macroporous, forming a "hamburger"-like structure.

[0102] 2.2 FTIR

[0103] Based on FTIR results ( Figure 3 C), the spectrum of DBNC shows that at 3342 cm -1 The peaks on the left and right are the stretching vibrations of hydroxyl (OH), at 292 cm -1 The peaks on the left and right are CH stretching vibrations, at 1020 cm -1 The CO stretching vibration peak of the hydroxyl group is at 1730 cm -1The peaks on the left and right are carbonyl (C=O) stretching vibrations at 880 cm -1 The peaks on the left and right are hemiacetal stretching vibrations, which all indicate that the C2 and C3 hydroxyl groups of BNC were successfully oxidized to aldehyde groups under the action of sodium periodate.

[0104] The spectrum of alginate is at 1608 cm -1 A peak appears at 1415 cm, which is due to the stretching of the carboxylate by the asymmetric extension of COO, indicating the content of aldehyde acid in the polymer. -1 The CO stretching band is attributed to the symmetrical stretching vibration of COO-. The stretching vibration of aliphatic CH appears at 2920-2850cm -1 In the infrared absorption curve of GelMA, the amide B band -CH asymmetric stretching causes 2933cm -1 The absorption peak at 1660 cm-1 is caused by the C=O stretching vibration of the amide I band. -1 The absorption peak at 1550cm is caused by the NH bending vibration of the amide I band. -1 The absorption peak at 1238 cm is caused by the -CH bending vibration of the amide III band. -1 There are absorption peaks at 3200 to 3400 cm -1 The band represents the presence of the peptide bond (NH stretching) of amide A, 3070 cm -1 The signal is the stretching vibration of the CH bond of amide B, which indicates the basic protein structure of GelMA. In the infrared spectrum of GDA generated by Schiff-base reaction of DBNC and GelMA, -CHO in DBNC is at 1720cm -1 The characteristic peak at 3400-3500 cm -1 The absorption peak in the range disappears, 1549cm -1 Nearby is the characteristic peak of -C=N imine bond in the product, indicating that -CHO in DBNC reacts with -NH in GelMA. At the same time, the characteristic absorption peaks of sodium alginate also exist.

[0105] 2.3 Swelling properties and drug release

[0106] like Figure 3As shown in B, when Alg is added to GD, its swelling performance is greatly reduced. Considering the change in its internal macroporous structure, the internal connection of the material is tighter, and this structure is more consistent with the internal structure of the growth plate cartilage; and the addition of calcium ions makes the internal connection of the hydrogel tighter, so its swelling rate at different time points is much smaller than that of the group without calcium ions, suggesting that Alg forms a denser hydrogel structure after cross-linking with calcium ions. Even so, its swelling rate can still reach about 500%. Interestingly, even if the material does not contain Alg, the addition of calcium ions can still reduce its swelling rate, which may be related to the cross-linking of the internal negative charge of DBNC with the positive charge of calcium ions.

[0107] Fig.16 What is shown is the release curve of IGF-1. It can be seen from the figure that there will be a burst release effect in the first three days or so, while the subsequent release is relatively stable. In addition, in the calcium ion-containing material group, the release of IGF-1 is significantly lower than that in the calcium-free group, indicating that the dense structure of the hydrogel after calcium cross-linking can release growth factors more slowly.

[0108] 3. In vitro experiments

[0109] 3.1 Biocompatibility Exploration

[0110] The biocompatibility of the hydrogel was tested by co-culture with Transwell plates. The live-dead staining results after 24h and 48h of co-culture of BMSCs and hydrogels showed that BMSCs cells grew well, no obvious dead cells appeared, and the cells were in good condition, indicating that the hydrogel had good cell compatibility and was suitable for direct cell culture ( Figure 4 Middle A). When BMSCs were co-cultured with hydrogels for 1, 3, and 5 days, CCK8 results showed that the cells proliferated well, regardless of the presence or absence of calcium ions. Figure 4 (middle B).

[0111] In order to explore the growth morphology of BMSCs on hydrogels, we conducted electron microscopy observations. Figure 3 As shown in A, BMSCs can be spread relatively completely on the hydrogel and grow well; when calcium ions are present, the cell morphology is different from that of the relatively soft matrix hydrogel, and a large number of calcium-phosphate nodules are secreted, indicating that the stem cells are transformed into osteoblasts.

[0112] 3.2 Exploration of differentiation induction ability

[0113] To verify that the hydrogel has a differentiation-directed effect under specific conditions, we incubated BMSCs with GDAI and GDAI+Ca 2+ After 7 days of co-culture, RNA-seq was performed and the differentially expressed genes were as follows: Figure 4As shown in A, B, C and D, we also performed GO analysis, KEGG analysis and String analysis on the differentially expressed genes.

[0114] like Figure 5 As shown in E and F, when BMSCs were co-cultured in the blank group and the GDAI group, the GO analysis of the differentially expressed genes in the BP aspect mainly included the cell response to insulin hormones and the activation of insulin pathways. At the same time, the IGF-1 receptor pathway also had significant differences, which indicated that the material group was able to release IGF-1 and exert its effects. The subsequent chondrocyte proliferation, development and differentiation also had significant differences. It is worth noting that the mTOR pathway and the BMP pathway were also activated. In the subcellular localization, it was mainly concentrated in the synthesis of protein kinases, methyltransferase complexes, insulin receptor complexes and transcriptional regulator complexes. In the molecular functions, multiple combinations were reflected, including GTP, DNA, proteoglycans, collagen, growth factors and IGF-1.

[0115] KEGG results Figure 5 As shown in F, the pluripotency of stem cells has obvious differences, which may indicate that under the action of growth factors, stem cells can be directed to differentiate; the PI3K-Akt-mTOR pathway is activated, suggesting that IGF-1 may exert its effect on chondrocytes through the above pathways, and the activation of the AMPK pathway, Notch pathway and TGF-β pathway is considered to be due to the fact that the "PI3K-Akt" signaling pathway is the downstream of multiple signal transduction processes, and its downstream also controls a variety of cell functions. It is worth noting that in the KEGG pathway, the HiF-1α signaling pathway is meaningful, and HiF-1α is a microenvironmental factor that is crucial for regulating chondrocyte differentiation.

[0116] GDAI+Ca 2+ After GO enrichment analysis of the differentially expressed genes between the group and the blank group ( Figure 6 A), we found that the biological process of downregulation was different from that of the GDAI group, and it was more focused on bone development, osteogenic differentiation and bone calcification, suggesting that calcium ions can promote the directional differentiation of BMSCs into osteoblasts, but its IGF-1 receptor pathway also exists, suggesting that calcium ions enhance the strength of the material at any time, but the IGF-1 inside it can still be released into the cells and play a role, while the activation of the MAPK pathway is considered to be related to osteogenic differentiation. Subcellular localization is mainly concentrated in chromosome condensation, intracellular skeleton formation and intercellular connection; molecular function suggests the existence of IGF-1 binding, calcium ion channel activation, ATP activity and DNA binding. KEGG results suggest ( Figure 6In B), the activated pathways include p53 pathway, TGF-β pathway, MAPK pathway and PI3K-Akt pathway, which may be related to the presence of calcium ions and the osteogenic differentiation of BMSCs. As for ferroptosis and apoptosis, it is considered that Ca 2+ is related to the continued existence of.

[0117] Subsequent String protein network interaction analysis suggested that the differentially expressed IGF-1R in the GDAI group was related to the PI3K-Akt signaling pathway, while Crebbp was related to the TGF-β signaling pathway, and Notch2 was an important protein involved in bone remodeling and balance ( Fig.17 Middle A); GDAI+Ca 2+ Map3k5 in the group suggests that it is related to the MAPK signaling pathway, while Pbk is related to the PI3K-Akt signaling pathway, which further verifies the results of KEGG ( Fig.17 (middle B).

[0118] To verify whether our hydrogel can reduce the formation of bone bridges in the presence of calcium ions, BMSCs were cultured in GD+Ca 2+ 、GDA+Ca 2+ and GDAI+Ca 2+ The material was cultured for 7 days and RT-PCR was performed. The results were as follows Figure 6 As shown in C, the expression of the marker osteogenic genes RUNX2, BMP2 and OST were significantly decreased in the GDAI group, indicating that under the action of IGF-1, even in the presence of calcium ions, the effect on the osteogenic differentiation of BMSCs was significantly reduced.

[0119] 4. In vivo experiments

[0120] 4.1MRI evaluation of growth plate regeneration in vivo

[0121] In order to verify the in vivo growth plate repair and regeneration ability of the composite hydrogel, we implanted hydrogels composed of different materials into the proximal tibial growth plate injury model of rats. During the repair process, the cross-linked hydrogels could remain in place and achieve the effect of sealing the wound ( Figure 7 (A).

[0122] Eight weeks after surgery, MRI was used to evaluate the growth plate repair. Figure 7 As shown in Figure B, from the coronal plane, the blank group had the largest bone bridge, followed by the GDA and GD groups, and the GDAI group had the smallest residual bone bridge, and the differences among the groups were statistically significant; from the sagittal plane, the length of the bone bridges among the blank group, GD group, and GDA group was not much different, but they were significantly different from the length of the bone bridges in the GDAI group; from the cross-sectional plane, the blank group had the largest bone bridge, which was much larger than that of the other groups and was statistically significant, while the GDAI group had the smallest bone bridge ( Figure 7 The above results indicate that the GDAI hydrogel we constructed can reduce the occurrence of bone bridges after growth plate injury in vivo.

[0123] 4.2 Immunohistochemical staining to evaluate growth plate regeneration

[0124] To further visualize the growth plate regeneration, we performed HE staining and TB staining. Figure 8 As can be seen in Figure A, HE staining successfully established a growth plate injury model. The formation of bone bridges was different among the groups, especially in the GDAI group, where the bone bridge formation was the smallest. The length of the bone bridge and the proportion of the growth plate were statistically analyzed. Figure 8 As shown in the results of B, the bone bridge in the GD group was the largest, followed by the blank group, and the smallest in the GDAI group, and the difference was statistically significant. TB staining was further performed, and it can be seen from the staining results that the bone bridge in the blank group was obvious, and the regeneration and connection of the growth plate in the other groups were visible. However, although the GD group and the GDA group were connected together, the color was lighter and the chondrocytes were arranged in disorder; however, the growth plates at the ends of the GDAI group were basically connected together, and the chondrocytes were arranged neatly ( Figure 8 ).

[0125] Immunohistochemical staining was performed on the tissue sections. Fig. 9 As can be seen in A, the blank group has obvious bone bridge formation, and its cartilage marker gene SOX9 is significantly reduced compared with other groups; all material groups have a large amount of SOX9 expression, including inside the growth plate. Among them, although the GD combination and GDI group have a large amount of SOX9 expression, only the GDAI group has uniform SOX9 inside the growth plate, while the other two groups have partial middle sections of the growth plate. Further staining is used to evaluate the degree of cartilage ossification after growth plate injury. Fig. 9 As can be seen in B, a large amount of Col II is expressed in the bone bridge of the blank group, while in the GD group, although Col II is less expressed, the cancellous bone underneath is also destroyed and the related Col II is also significantly reduced, while the GDA and GDAI groups have less Col II expression and the growth plate is uniform. Hif-1α is an important microenvironmental factor for cartilage regeneration. Fig. 9 As shown in middle C, except for the bone bridge part, there was basically no expression between the growth plates in the blank group. However, in the material group, Hif-1α was uniformly expressed, indicating that the implantation of materials at the damaged site can block it and create a hypoxic microenvironment, which is conducive to the regeneration and repair of the growth plate.

[0126] 4.3 RNA-seq detection of the role of materials in vivo

[0127] To further examine the process of the material's function in vivo, we performed RNA-seq on the blank group and the GDAI material group ( Fig.10 (A) The differentially expressed genes between the two groups are as follows Fig.10 As shown in B, it can be seen that the genes related to cartilage regeneration are significantly increased.

[0128] Subsequent GO analysis showed that the biological process mainly involved changes in the skeletal system, including cartilage differentiation, endogenous cartilage growth, bone growth, growth plate development, etc., as well as MAPK activation and VEGF signaling pathway activation; subcellular localization was in the intracellular skeleton, mitochondria, ribosomes, and calcium channel complexes; molecular functions were ATP activation, ribosome assembly, calmodulin binding, growth factor binding, ion channel binding, etc. ( Fig.10 Subsequent KEGG analysis showed ( Fig.11 In Figure A), the main activated pathways include: PI3K-Akt signaling pathway, calcium ion pathway, growth hormone-related pathway, MAPK pathway, AMPK pathway, Notch pathway, oxidative phosphorylation and mineral deposition pathway, which are basically consistent with the pathways activated in cell experiments. String analysis also further suggests that PI3K-Akt and related signaling pathways are activated ( Fig.11 In addition, the hydrogel injected into the damaged area can seal the wound, create a hypoxic microenvironment, and promote cartilage regeneration through the HIF-1α signaling pathway ( Fig.11 (middle B).

[0129] According to the main expressed genes in each growth plate partition reported in the literature, and compared with our RNA-seq differentially expressed genes ( Fig.12 A), the result is as follows Fig.12 As shown in B, the bold italics are significantly up-regulated differentially expressed genes, and the underlined ones are significantly down-regulated differentially expressed genes. As can be seen from the figure, from the resting zone to the hypertrophic zone, the up-regulated genes account for the majority, while there are more down-regulated genes in the calcified zone, indicating that the implanted material can better activate the gene expression of the resting layer, the proliferation layer and the hypertrophic layer of the growth plate, and can also inhibit the osteogenic gene expression of the calcified layer. We performed RT-PCR detection on KLF13, RUNX3, BMP2 and COL10A1, and the results showed that the gene expression in the GDAI group was significantly increased compared with the blank group, indicating that the experimental group can better promote the regeneration and repair of the growth plate cartilage ( Fig.12 Middle C).

[0130] In summary, the new hydrogel material constructed by the present invention can promote the chondrogenic differentiation of BMSCs due to its soft matrix characteristics in the absence of calcium ions, especially after the addition of IGF-1, it can activate the chondrogenic differentiation signaling pathways of related BMSCs, such as the PI3K-AKT signaling pathway and the TGF-β signaling pathway. When calcium ions are present, as the culture time increases, the soft matrix characteristics of the hydrogel gradually transform into a hard matrix, which can promote the formation of the cytoskeleton and microtubules of BMSCs, and may play an important role in the subsequent osteogenic differentiation. However, the hydrogel material can still release IGF-1 and play a role, reducing the expression of related osteogenic genes and avoiding the formation of bone bridges.

[0131] Therefore, the present invention constructs a novel double-network hydrogel scaffold, using the amino group of GelMA and the aldehyde group of DBNC to form the first layer of network through Schiff base bond connection, and the Alg inside the material can realize the self-reinforcement of the hydrogel by binding to the calcium ions in the tissue, and the matrix of the material close to the bone is gradually enhanced, while the part close to the growth plate cartilage maintains the strength of the soft matrix, forming a "hamburger"-like structure, so that it can match the pathological state after the growth plate injury, thereby promoting the regeneration of the growth plate cartilage. After this adaptive hydrogel scaffold is injected into the growth plate injury area, it can quickly gel, and then block the wound, creating a suitable hypoxic microenvironment for cartilage regeneration. More importantly, on this basis, IGF-1 can be slowly released, and further promote the regeneration and repair of the growth plate cartilage by activating the PI3K-Akt signaling pathway. In short, this novel hydrogel scaffold provides a new idea for the regenerative application of clinical treatment of diseases related to growth plate injury in children.

Claims

1. A method for preparing an injectable double-network hydrogel scaffold, characterized in that: The following steps are involved: (1) dissolving bacterial cellulose, adding an oxidant NaIO4 to carry out an oxidation reaction, and preparing oxidized bacterial cellulose; (2) preparing a mixed solution of methacrylic anhydride gelatin hydrogel solution, oxidized bacterial cellulose and sodium alginate, and performing photo-crosslinking and curing to prepare a double network hydrogel; (3) Dissolving the double network hydrogel obtained in step (2) in water, and then mixing it with the growth factor IGF-1 to prepare a double network hydrogel scaffold loaded with IGF-1.

2. The method according to claim 1, characterized in that The weight ratio of bacterial cellulose to NaIO4 in step (1) is 2:

1.

3. The method according to claim 1, characterized in that The temperature of the oxidation reaction in step (1) is 40° C., the reaction time is 14 h, and the reaction pH is 6.

0.

4. The method according to claim 1, characterized in that The method for preparing the methacrylic anhydride gelatin hydrogel in step (2) is to disperse gelatin in a carbonate buffer solution, then react with methyl acrylate, and prepare the methacrylic anhydride gelatin hydrogel by centrifugal dialysis.

5. The method according to claim 1, characterized in that The weight ratio of the methacrylic anhydride gelatin hydrogel solution, oxidized bacterial cellulose and sodium alginate in step (2) is 7.5:1:1.

2.

6. The method according to claim 1, characterized in that The amount of IGF-1 added in step (3) is 100 ng / mL.

7. An injectable double-network hydrogel scaffold prepared by the method according to any one of claims 1 to 6.

8. The injectable double-network hydrogel scaffold according to claim 7, characterized in that: The double-network hydrogel scaffold has the ability to autonomously recruit calcium ions in vivo.

9. Use of the injectable double-network hydrogel scaffold according to claim 7 or 8 in preparing an implant material for treating growth plate cartilage damage.

10. Use of the injectable double-network hydrogel scaffold according to claim 7 or 8 in the preparation of a drug for treating growth plate cartilage damage.

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