Injectable temperature-sensitive hydrogel as well as preparation method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- OCEAN UNIV OF CHINA
- Filing Date
- 2026-01-23
- Publication Date
- 2026-04-21
Smart Images
Figure CN121895643A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of hydrogel preparation technology, specifically to an injectable thermosensitive hydrogel (HBCS / MPG) based on a composite of hydroxybutyl chitosan (HBCS) and melanin-polyethylene glycol / gelatin core-shell microspheres (MPG), its preparation method, and its application. Background Technology
[0002] The clinical treatment of intervertebral disc degeneration has long faced significant challenges, including the irreversible degenerative process and the imbalance between invasive interventions and the need for tissue repair. Traditional treatment methods are limited by multiple bottlenecks, such as surgical trauma, implant biomechanical mismatch, and the continuous deterioration of the inflammatory microenvironment, making it difficult to achieve dual structural and functional repair. Injectable thermosensitive hydrogel technology, based on minimally invasive delivery, rapidly constructs a biomimetic three-dimensional scaffold by responding to body temperature with intelligent materials. Its dynamic mechanical properties are highly matched with the viscoelasticity of the natural intervertebral disc, forming a stable mechanical support system. Simultaneously, this system targets and integrates antioxidant and immunomodulatory functions, effectively inhibiting oxidative stress and the inflammatory cascade, reshaping the local microenvironment homeostasis, and stimulating the repair potential of nucleus pulposus cells and the matrix regeneration capacity. Summary of the Invention
[0003] The purpose of this invention is to provide an injectable thermosensitive hydrogel based on a composite of hydroxybutyl chitosan and melanin-polyethylene glycol / gelatin core-shell microspheres, its preparation method, and its application, so as to overcome the shortcomings of the prior art.
[0004] Hydroxybutyl chitosan (HBC) exhibits excellent water solubility, overcoming the limitation of poor water solubility in chitosan and facilitating drug mixing and injection. Its thermosensitivity allows it to rapidly form a gel near body temperature, enabling in-situ drug release and prolonging the duration of drug action. This material demonstrates good biocompatibility, lack of cytotoxicity, and high safety; its biodegradability ensures that degradation products are harmless to the body. Combining HBC with melanin to form a hydrogel can improve its mechanical and antibacterial properties. However, melanin alone has poor solubility and is not readily reactive. Therefore, polyethylene glycol is introduced to improve its solubility and the activity of reactive groups. Since all the above materials are chemically synthesized, the resulting hydrogels exhibit poor biocompatibility. Therefore, gelatin is introduced into the hydrogel to improve biocompatibility and reduce immunogenicity.
[0005] Melanin contains catechols, which can induce cells to produce antioxidants, maintain oxygen balance by scavenging reactive oxygen species, and combat oxidative stress damage. Coupling diamino polyethylene glycol (PEG-(NH2)2) to melanin enhances its water solubility and prolongs its in vivo half-life. Its metal ion chelating ability can remove excess metals from pathological environments, reducing oxidative stress damage; its nanoscale size enhances penetration into diseased tissues, promoting targeted drug delivery. Gelatin, as a natural polymer, is absorbable by the human body without immune rejection; its outstanding gelling properties allow it to form a stable gel network, which is beneficial for cell adhesion and proliferation; its good film-forming properties allow it to form a protective film on the material surface, regulating drug release; its excellent water-holding properties maintain the material's hydration state and preserve tissue physiological function. Medical materials for treating nucleus pulposus degeneration are prepared by chemically synthesizing and characterizing the raw materials.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] An injectable thermosensitive hydrogel (HBCS / MPG) based on a composite of hydroxybutyl chitosan (HBCS) and melanin-polyethylene glycol / gelatin core-shell microspheres (MPG) is disclosed. The hydrogel is composed of a mixture of hydroxybutyl chitosan (HBCS) and melanin-polyethylene glycol / gelatin core-shell microspheres (MPG); the mass ratio of HBCS to MPG is 1-27:1-4.
[0008] The properties of the injectable thermosensitive hydrogel (HBCS / MPG) are as follows: the hydrogel has a porous network structure with uniform pore distribution, a compact structure, and small pore size; the storage modulus G' is higher than the loss modulus G''; it has a high swelling ratio; it has good stability, and the hydrogel can remain stable at body temperature of 37°C or even higher; the degradation rate of the hydrogel can reach about 40% at an enzyme concentration of 8000 U; the degradation rate is about 20% at an enzyme concentration of 2000 U; and the HBCS / MPG hydrogel has good antioxidant properties.
[0009] The preparation method of the injectable thermosensitive hydrogel (HBCS / MPG) includes the following steps: (1) Preparation of hydroxybutyl chitosan HBCS; (2) Preparation of melanin-polyethylene glycol / gelatin core-shell microspheres (MPG); (3) Dissolve the prepared HBCS and MPG in deionized water, stir thoroughly, and then mix the HBCS solution and MPG solution. The mass ratio of HBCS and MPG is 1-27:1-4. The mixture is ready to form a gel.
[0010] Further, step (1) specifically involves: adding chitosan to distilled water and stirring until well mixed, then adding acetic acid. After complete dissolution, slowly adding NaOH until alkaline, and stirring until a latex-like precipitate is obtained. Adding the white precipitate to isopropanol and water, heating and increasing the rotation speed, and slowly adding 1,2-epoxybutane to continue the reaction. After the reaction is completed, cooling to room temperature, dialyzing until the ionic strength of the dialysate is 0, centrifuging, and freezing-drying the supernatant to obtain HBCS.
[0011] Furthermore, step (2) includes: 1) Dissolve NH2-PEG-NH2 and melanin separately in Tris buffer and stir until completely dissolved; then weigh EDC and NHS and add them to the above solution, followed by PEG solution. After the reaction is complete, remove impurities with anhydrous ethanol, wash the precipitate three times, dialyze and freeze dry to obtain MP product. 2) Weigh gelatin and add it to MES buffer for hydration until the solution is clear and transparent; then add EDC and NHS to the solution and stir at room temperature until completely dissolved; weigh MP and dissolve it in Tris solution and stir overnight until dissolved; mix the two reaction solutions, react at room temperature, dialyze, and freeze dry to obtain MPG.
[0012] Furthermore, in step (3), the prepared HBCS is dissolved in deionized water to prepare solutions with concentrations of 2% and 3% (w / v), and the prepared MPG is dissolved in deionized water to prepare solutions with concentrations of 2%, 3%, and 4% (w / v). After thorough mixing, the HBCS solutions and MPG solutions of different concentrations are mixed in different proportions until a gel is formed.
[0013] Application of the injectable thermosensitive hydrogel (HBCS / MPG) in the preparation of anti-inflammatory products.
[0014] Application of the injectable thermosensitive hydrogel (HBCS / MPG) mixed with rat adipose mesenchymal stem cell exosomes ADSCs-exo in the preparation of anti-inflammatory products.
[0015] Compared with the prior art, the present invention has at least the following beneficial effects:
[0016] 1. A biomimetic hydrogel based on a hydroxybutyl chitosan (HBCS) microporous network and melanin / polyethylene glycol (MPG) core-shell microspheres was developed, matching the structure and function of the nucleus pulposus tissue. This system constructs a three-dimensional porous network based on hydroxybutyl chitosan, precisely matching the structure of natural nucleus pulposus tissue. The surface charge properties of the material are combined to optimize the cell adhesion microenvironment. Simultaneously, core-shell composite microspheres are integrated, endowing the material with antioxidant functions and controlled drug release capabilities. Through the thermosensitive gel properties, a mechanical buffer barrier is rapidly formed, creating a biomimetic hydration network, ultimately achieving dynamic adaptation to the biomechanical environment of the intervertebral disc.
[0017] 2. An innovative dual-signal cascade regulatory mechanism of "antioxidant and anti-inflammatory-regenerative synergy" overcomes the limitations of traditional single-function materials. It enables dynamic scavenging of oxidative stress: melanin in MPG directly captures ROS through polyphenolic groups, and in conjunction with HBCS amino groups, inhibits oxidase activity, significantly reducing intracellular ROS levels; it also enables precise remodeling of the inflammatory microenvironment: loaded exosomes target and inhibit the NF-κB pathway via miRNA-140, downregulating IL-6 / IL-1β while simultaneously upregulating IL-10, achieving inflammatory reversal and immune homeostasis reconstruction. Attached Figure Description
[0018] Figure 1 (I) Infrared spectra of chitosan, hydroxybutyl chitosan (A), melanin-polyethylene glycol, and melanin-polyethylene glycol-gelatin (B); (II) Nuclear magnetic resonance spectra of hydroxybutyl chitosan (A) and melanin-polyethylene glycol-gelatin (B); (III) Scanning electron micrographs of hydroxybutyl chitosan, melanin-polyethylene glycol, and melanin-polyethylene glycol-gelatin (from left to right).
[0019] Figure 2 (I) Scanning electron micrograph of HBCS3 / MPG4 hydrogel; (II) Frequency scan of modulus of HBCS2 / MPG2 (A), HBCS2 / MPG3 (B), HBCS2 / MPG4 (C), HBCS3 / MPG3 (D), and HBCS3 / MPG4 (E) hydrogels; (III) Strain scan of modulus of HBCS2 / MPG2 (A), HBCS2 / MPG3 (B), HBCS2 / MPG4 (C), HBCS3 / MPG3 (D), and HBCS3 / MPG4 (E) hydrogels.
[0020] Figure 3 (I) Swelling ratio of HBCS / MPG hydrogels with different ratios; (II) Stability of HBCS / MPG hydrogels with different ratios; (III) Thermogravimetric analysis of HBCS2 / MPG2 (A), HBCS2 / MPG3 (B), HBCS2 / MPG4 (C), HBCS3 / MPG3 (D), and HBCS3 / MPG4 (E) hydrogels; (IV) Degradation curves of HBCS / MPG hydrogels with different ratios at enzyme concentrations of 2000 U (A), 5000 U (B), and 8000 U (C) over time.
[0021] Figure 4 The DPPH scavenging ability of HBCS / MPG hydrogels with different ratios was investigated.
[0022] Figure 5(I) The viability of L929 cells and human nucleus pulposus cells after co-culturing with different hydrogel solutions for 24, 48, and 72 hours; (II) The growth status of L929 cells and human nucleus pulposus cells after co-culturing with HBCS3 / MPG4 and HBCS3 / MPG4+Exo hydrogel solutions for 24, 48, and 72 hours.
[0023] Figure 6 (I) The three-dimensional migration of L929 cells (A) and human nucleus pulposus cells (B) after co-culturing with hydrogel solution for 24 h, and the number of L929 cells (C) and human nucleus pulposus cells (D) that migrated (n=6, *p<0.05, **p<0.01, ***p<0.001); (II) The three-dimensional migration of L929 cells (A) and the number of migrating cells (B) after co-culturing with HBCS3 / MPG4 hydrogel and HBCS3 / MPG4+Exo hydrogel for 24 h.
[0024] Figure 7 The purpose was to (I) evaluate the ROS scavenging capacity of hydrogels and their antioxidant effects at the cellular level using DCFH-DA fluorescence staining method; (II) evaluate the effect of different concentrations of H2O2 solution on the viability of human nucleus pulposus cells (A); and evaluate the protective effect of hydrogels on H2O2-stimulated human nucleus pulposus cells (B) (*p<0.05, **p<0.01, ***p<0.001).
[0025] Figure 8 (I) Subcutaneous injection (A) and intramuscular injection (B) of HBCS3 / MPG4; (II) HE staining of HBCS3 / MPG4; (III) Hemolysis rate of HBCS3 / MPG4 (n=3, *p<0.05, **p<0.01, ***p<0.001).
[0026] Figure 9 Magnetic resonance imaging (A) and water content of the intervertebral disc (B).
[0027] Figure 10 HE staining of the intervertebral disc.
[0028] Figure 11 Masson staining for intervertebral discs.
[0029] Figure 12 Immunofluorescence of IL-6 in the intervertebral disc. Detailed Implementation
[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description, in conjunction with specific embodiments and accompanying drawings, further illustrates the invention. Obviously, the described embodiments are only a portion, not all, of the embodiments disclosed in this invention. All other embodiments obtained by those skilled in the art based on the embodiments disclosed in this invention without inventive effort are within the scope of protection of this invention.
[0031] Example 1
[0032] Preparation of HBCS: Take 5 g of chitosan, add it to distilled water and stir until well mixed. Then add acetic acid and slowly add NaOH until alkaline. Stir until a latex-like precipitate is obtained. Place the white precipitate in a three-necked flask, add isopropanol and water, stir at room temperature for 12-24 h, slowly add 1,2-epoxybutane and continue the reaction for 24-48 h. Dialyze until the ionic strength of the dialysate is 0, take it out, centrifuge and freeze-dry the supernatant to obtain HBCS.
[0033] MPG preparation process:
[0034] (1) Dissolve NH2-PEG-NH2 and melanin separately in Tris buffer and stir until completely dissolved. Then weigh EDC and NHS and add them to the above solution, followed by PEG solution and react for 24-48 h. After the reaction, remove impurities with anhydrous ethanol, wash the precipitate three times, dialyze and freeze dry to obtain MP product.
[0035] (2) Weigh the gelatin and add it to the MES buffer. Then add EDC and NHS to the solution and stir at room temperature until completely dissolved. Add MP and react at room temperature for 24-48 h. Then dialyze and freeze dry to obtain MPG.
[0036] Experiments to determine the properties of HBCS and MPG: 1. Dissolve HBCS in water, incubate overnight at low temperature, form a gel in a water bath, then rapidly freeze with liquid nitrogen, followed by freeze-drying. Spray gold onto the cross-section and observe the internal morphology using a scanning electron microscope (SEM). Separately weigh MP and MPG samples and dissolve them to prepare solutions. Stir overnight to dissolve and fully disperse the solid in water. Then, filter the nanoparticles through a 0.22 μm filter membrane and observe their morphology using SEM.
[0037] 2. The infrared absorption spectra of HBCS, MP, and MPG were acquired using a Fourier transform infrared spectrometer, and the proton NMR spectra of HBCS, MP, and MPG were obtained by nuclear magnetic resonance characterization. The chemical structures of HBCS, MP, and MPG were then investigated.
[0038] Results analysis: 1. For example Figure 1 As shown, Figure 1 - (Ⅰ) are the Fourier transform infrared spectra of CS, HBCS (A) and MP, MPG (B). From Figure (A), it can be seen that the chemically modified products have a wavelength of 3434 cm⁻¹. -1 Nearby, hydroxyl and ammonia are characterized at 1085 cm⁻¹ -1 The CO single bond stretching vibration shifts to a lower wavenumber and exhibits a superposition effect with adjacent vibrational modes, indicating that the C6 hydroxyl group in the sugar ring participates in the etherification crosslinking reaction, thus proving the successful preparation of HBCS. As shown in Figure (B), in the spectrum, at 3385 cm⁻¹... -1 The amino (NH) stretching vibration peak at 2926 cm⁻¹ indicates the presence of amino groups in MPG, possibly originating from diamino polyethylene glycol or gelatin. -1 The methyl (CH) stretching vibration peak at 1466 cm⁻¹ -1 The CH bending vibration peak at 1628 cm⁻¹ indicates the presence of polyethylene glycol chains. -1 The carbonyl (C=O) stretching vibration peak at 1045 cm⁻¹ likely originates from peptide bonds in gelatin or oxide groups in polyethylene glycol. -1 The stretching vibration peak of the ether bond (CO) at the position further confirmed the successful synthesis of MPG.
[0039] 2. The results of the 1H NMR analysis of HBCS are as follows: Figure 1 As shown in (A) of (II), the chemical shifts are: 0.84 ppm for the methyl group, 1.85 ppm for the methylene group of the pyran ring, 2.85 ppm for the methine group attached to the amino group, 3.62 ppm for the methylene group, 7.80 ppm for the hydroxyl group, and 8.40 ppm for the amino group. In summary, HBCS was successfully prepared. The 1H-NMR spectrum analysis in (B) provides strong evidence for the successful synthesis of MPG. The NH peak of the amide bond at 8.4 ppm indicates that a chemical bond was successfully formed between melanin and PEG, a key step in MPG synthesis. The peaks at 3.9 ppm and 3.4 ppm further confirm the presence of the PEG structure, indicating that PEG has been successfully grafted onto melanin. Furthermore, the peaks at 0.9 ppm, 1.4 ppm, 2.7 ppm, and 3.6 ppm correspond to characteristic groups of different amino acids, indicating that the protein components in gelatin are retained in MPG.
[0040] 3. Figure 1-(Ⅲ) Scanning electron microscope images of HBCS, MP, and MPG from left to right. HBCS exhibits a honeycomb-like three-dimensional porous network structure. The pore walls of this structure are relatively smooth and dense, with large pore sizes, and the network structure is relatively loose and not very smooth. MP nanoparticles are uniform in size and have clear edges, with particle sizes mainly distributed between 100-300 nm, and they tend to aggregate. MPG nanoparticles are uniform in size and have clear edges, with particle sizes mainly distributed between 100-200 nm, and they tend to aggregate.
[0041] Example 2:
[0042] Preparation and gelation experiments of injectable thermosensitive hydrogel system (HBCS / MPG): The gelation temperature and time of the hydrogel were determined using the inverted vial method. HBCS was dissolved in deionized water to prepare solutions with concentrations of 2% and 3% (w / v). MPG was also dissolved in deionized water to prepare solutions with concentrations of 2%, 3%, and 4% (w / v). After thorough mixing, the HBCS and MPG solutions of different concentrations were mixed in different ratios (1:9; 3:7; 1:1; 7:3; 9:1). The hydrogels of 2% HBCS and 2% MPG were abbreviated as HBCS2 / MPG2, 2% HBCS and 3% MPG as HBCS2 / MPG3, 2% HBCS and 4% MPG as HBCS2 / MPG4, 3% HBCS and 3% MPG as HBCS3 / MPG3, and 3% HBCS and 4% MPG as HBCS3 / MPG4. Each mixed solution was prepared in batches of 1... mL, immerse the glass bottle containing the solution in water baths at different temperatures at the initial temperature, and test the gelation time of the hydrogel at each temperature. That is, place it in the water bath for a certain time, take it out and invert the glass bottle. If no solution flows down the wall within 1 minute, it is considered that the hydrogel has been formed, and record the time.
[0043] After a water bath at 37°C for 3 minutes, HBCS / MPG mixtures at all volume ratios of 9:1 and 7:3 gelled. At a volume ratio of 1:1, only mixtures of HBCS2 and MPG2, and HBCS3 and MPG3, gelled, becoming non-flowing solids. All other concentrations and volume ratios failed to gel at 37°C.
[0044] The above experiments were conducted to determine the properties of the gel-forming HBCS / MPG composite hydrogel: 1. Structural observation of HBCS3 / MPG4 (volume ratio 7:3) composite hydrogel
[0045] After the HBCS / MPG hydrogel was gelled, it was freeze-dried, cut to obtain a cross-section, and the microstructure inside the hydrogel was observed using SEM.
[0046] 2. Rheological properties of HBCS / MPG hydrogel
[0047] The selected gel-forming solutions were mixed at a volume ratio of 7:3, with 2 mL of each of the following ratios: 2% HBCS and 2% MPG, 2% HBCS and 3% MPG, 2% HBCS and 4% MPG, 3% HBC and 3% MPG, and 3% HBC and 4% MPG. Oscillation frequency scanning and oscillation strain scanning were performed on the hydrogels with different ratios to investigate their structural stability, reversible deformation, and stability.
[0048] 3. Swelling properties of HBCS / MPG hydrogel
[0049] The selected gel-forming solutions were mixed at a volume ratio of 7:3: 2% HBCS and 2% MPG, 2% HBCS and 3% MPG, 2% HBCS and 4% MPG, 3% HBC and 3% MPG, and 3% HBC and 4% MPG. The hydrogels of different components were weighed and then immersed in PBS buffer (pH = 7.4). The mixtures were placed in a 37°C incubator, and the samples were removed, dried, and weighed at different time points until the hydrogels reached swelling equilibrium. The swelling rate of the hydrogels was then calculated.
[0050] (1-1)
[0051] In the formula, Ws is the mass of the hydrogel after swelling, and Wd is the mass of the original hydrogel, in g.
[0052] 4. Stability of HBCS / MPG hydrogel
[0053] The selected gel-forming solutions were mixed at a volume ratio of 7:3: 2% HBCS and 2% MPG, 2% HBCS and 3% MPG, 2% HBCS and 4% MPG, 3% HBBC and 3% MPG, and 3% HBBC and 4% MPG. The prepared HBCS / MPG hydrogel samples were dried until a constant mass was achieved; the mass of the dried hydrogel was recorded as M0. The dried hydrogel samples were then completely immersed in an appropriate amount of PBS solution, ensuring the hydrogel was fully submerged. They were placed in a 37°C incubator to simulate human body temperature. The PBS solution was changed daily, and the hydrogel sample was removed every other day. Excess solution was gently wiped off the surface of the hydrogel with filter paper, and the mass of the hydrogel was measured and recorded as Mt. The time point of each weighing and the corresponding hydrogel mass were recorded. The hydrogel mass Mt obtained from each weighing was compared with the initial dried mass M0 to calculate the mass change rate or residual rate of the hydrogel. By plotting the mass change curve, the stability of the hydrogel in PBS solution over time can be visually observed.
[0054] 5. Thermal stability of HBCS / MPG hydrogel
[0055] The selected gel-forming solutions were mixed at a volume ratio of 7:3: 2% HBCS and 2% MPG, 2% HBCS and 3% MPG, 2% HBCS and 4% MPG, 3% HBC and 3% MPG, and 3% HBC and 4% MPG. The lyophilized samples of HBCS / MPG hydrogels with different ratios were ground into powder and placed in a thermogravimetric analysis crucible. The mass change of the gel samples was determined using a thermogravimetric analyzer. Under air atmosphere, the samples were placed in an alumina crucible, and experiments were conducted at a temperature range of 25℃-600℃ with a heating rate of 10°C / min.
[0056] 6. Degradation properties of HBCS / MPG hydrogel
[0057] The selected gel-forming solutions were mixed at a volume ratio of 7:3: 2% HBCS and 2% MPG, 2% HBCS and 3% MPG, 2% HBCS and 4% MPG, 3% HBC and 3% MPG, and 3% HBC and 4% MPG, respectively. The hydrogels were then placed in 2000 U, 5000 U, and 8000 U lysozyme solutions and incubated in a shaker at 37 °C. The lysozyme solution was replaced at fixed intervals, and the samples were weighed. The enzyme degradation rate was calculated. Weight loss rate (%) = ×100% (1-2)
[0058] In the formula, 𝑊0 is the initial mass of the sample; 𝑊𝑡 is the mass of the sample after degradation for time t.
[0059] 7. Antioxidant properties of HBCS / MPG hydrogel
[0060] The selected gel-forming solutions were mixed at a volume ratio of 7:3: 2% HBCS and 2% MPG, 2% HBCS and 3% MPG, 2% HBCS and 4% MPG, 3% HBC and 3% MPG, and 3% HBC and 4% MPG, respectively. DPPH working solutions were then prepared and stored at 2℃-8℃ for later use. Different hydrogels were added to the DPPH working solutions, and blank and control groups were set up. After the reaction was carried out in the dark, the absorbance was measured at 515 nm, and the DPPH free radical scavenging rate was calculated. (1-3)
[0061] In the formula, A1 is the OD value of the experimental group, A2 is the OD value of the blank group, and A0 is the OD value of the control group.
[0062] Experimental results:
[0063] 1. Figure 2 (Ⅰ) are scanning electron microscope images of HBCS3 / MPG4 hydrogels at different scales. (A) is 4 μm and (B) is 2 μm. It can be seen that the hydrogels all have a porous network structure with uniform pore distribution and a compact structure with small pore size. The higher the degree of cross-linking of the hydrogel, the more cross-linking points will be formed inside, which will lead to a decrease in the porosity and pore size of the hydrogel. Therefore, it can be seen that the compact network structure of HBCS / MPG hydrogel has better stability and mechanical strength.
[0064] 2. Figure 2(II) shows the frequency scanning results of the modulus of HBCS2 / MPG2 (A), HBCS2 / MPG3 (B), HBCS2 / MPG4 (C), HBCS3 / MPG3 (D), and HBCS3 / MPG4 (E) hydrogels. As can be seen from the figure, the storage modulus G' of the five hydrogels is higher than the loss modulus G'' within a certain frequency range, indicating that the HBCS / MPG hydrogels are all in a good gel state. Among them, the storage modulus G' of HBCS3 / MPG3 and HBCS3 / MPG4 hydrogels is much higher than the loss modulus G'', which indicates that the crosslinking degree of HBCS3 / MPG3 and HBCS3 / MPG4 hydrogels is higher than that of other concentration ratio hydrogels, and their network structure is more stable than that of other concentration ratio hydrogels. Furthermore, the storage modulus G' of HBCS2 / MPG4, HBCS3 / MPG3, and HBCS3 / MPG4 hydrogels were all higher than 100, with maximum values reaching 347.726, 184.701, and 225.306 Pa, respectively. The G' values were significantly higher than those of HBCS2 / MPG2 and HBCS2 / MPG3 hydrogels, indicating that the addition of melanin and hydroxybutyl chitosan made the hydrogel network more compact, resulting in higher mechanical properties and stability, which is beneficial for tissue recovery. Simultaneously, the modulus changes of the hydrogels under different stress-strain conditions were measured. Figure 2 (III) shows the strain scanning results of the hydrogel modulus of HBCS2 / MPG2 (A), HBCS2 / MPG3 (B), HBCS2 / MPG4 (C), HBCS3 / MPG3 (D), and HBCS3 / MPG4 (E). It can be clearly seen from the figure that in the strain range of 1%-100%, the storage modulus G' of HBCS2 / MPG2, HBCS2 / MPG3, HBCS2 / MPG4, HBCS3 / MPG3, and HBCS3 / MPG4 hydrogels is higher than the loss modulus G''. Moreover, compared with HBCS2 / MPG2, HBCS2 / MPG3, and HBCS2 / MPG4, the LVR region of HBCS3 / MPG3 and HBCS3 / MPG4 hydrogels is wider. The results indicate that HBCS3 / MPG3 and HBCS3 / MPG4 hydrogels have higher crosslinking degree, greater gel strength, and more stable structure.
[0065] 3. For example Figure 3As shown in (I), after 24 h of soaking, the swelling ratios of HBCS2 / MPG2, HBCS2 / MPG3, HBCS2 / MPG4, HBCS3 / MPG3, and HBCS3 / MPG4 hydrogels were 2.72, 2.19, 2.72, 3.23, and 3.26, respectively. The swelling ratios of HBCS3 / MPG3 and HBCS3 / MPG4 hydrogels were significantly higher than those of the other ratios, and within a suitable range. This indicates that HBCS3 / MPG3 and HBCS3 / MPG4 hydrogels can absorb a large amount of water, which helps restore the water content of the intervertebral disc, thereby maintaining the height and elasticity of the intervertebral disc. Furthermore, hydrogels with strong swelling capacity can alleviate the inflammatory response within the intervertebral disc, promote the regeneration of the extracellular matrix, provide a suitable microenvironment for intervertebral disc cells, promote cell proliferation and differentiation, enabling them to effectively load and release drugs, achieve sustained drug release, and further enhance the therapeutic effect.
[0066] 4. Place the hydrogel in a neutral buffer solution and weigh it after it has absorbed water and stabilized. Weigh the hydrogel at the same time every day thereafter and record the weight. The stability of the hydrogel after it enters the body will affect its duration of action in vivo. Figure 3 As shown in (II), the HBCS2 / MPG2, HBCS2 / MPG3, HBCS2 / MPG4, HBCS3 / MPG3, and HBCS3 / MPG4 hydrogels are all relatively stable. After soaking in PBS buffer (pH=7.4) for one week, their weights did not change significantly. Among them, HBCS2 / MPG3 has the best stability, while HBCS3 / MPG3 and HBCS3 / MPG4 have slightly worse stability, but all are within acceptable ranges.
[0067] 5. Thermogravimetric analysis (TGA) is primarily used in hydrogel research to assess its thermal stability. TGA determines the mass loss of hydrogels at different temperatures, thus revealing their thermal decomposition temperature and thermal stability. For example... Figure 3 As shown in (III), the mass loss of the hydrogel sample mainly occurs in two stages. The first stage is between 200-300℃, which is the stage of loss of the main components. Both diamino polyethylene glycol and gelatin are easily lost due to heat in this temperature range, and most of HBCS also degrades in this stage. The second stage is between 300-550℃, which is the stage of pyrolysis of the remaining HBCS residue. After further heating, at high temperatures of 550-800℃, some remaining substances still exist stably. This is because the melanin in the hydrogel is a high molecular weight compound formed by the complex polymerization reaction of multiple phenylalanine units. Its molecular structure contains a large number of stable structures such as conjugated double bonds and phenolic hydroxyl groups. These structures endow melanin with high thermal stability. Therefore, it can be seen that the hydrogel can remain stable at body temperature of 37℃ or even higher.
[0068] 6. Observe the degradation of the hydrogel in the presence of lysozyme. Figure 3 (IV) is a graph showing the in vitro degradation rate versus degradation time of the hydrogel at three lysozyme concentrations of 2000 U (A), 5000 U (B), and 8000 U (C). Figure 3 (IV) The degradation of HBCS2 / MPG2, HBCS2 / MPG3, HBCS2 / MPG4, HBCS3 / MPG3, and HBCS3 / MPG4 hydrogels in the presence of lysozyme, respectively. It can be seen that the hydrogels can be degraded under the action of the enzyme, and the degradation accelerates with increasing enzyme concentration. At an enzyme concentration of 8000 U, the degradation rate of the hydrogels can reach approximately 40%; at an enzyme concentration of 2000 U, the degradation rate is approximately 20%.
[0069] 7. The antioxidant activity of HBCS / MPG hydrogel was tested by measuring its DPPH radical scavenging ability. DPPH radicals have unpaired electrons, are purple in color, and have an absorption peak at 517 nm. The antioxidant components in the hydrogel can donate electrons or hydrogen atoms to DPPH radicals, converting them into colorless DPPH molecules, resulting in a lighter solution color and reduced absorbance. The antioxidant performance of the hydrogel can be evaluated by measuring the change in absorbance. Figure 4 As shown, the DPPH scavenging rates of HBCS2 / MPG2, HBCS2 / MPG3, HBCS2 / MPG4, HBCS3 / MPG3, and HBCS3 / MPG4 hydrogels were 78%, 82.94%, 80.514%, 55.66%, and 67.79%, respectively. Figure 4 The results show that 3% HBCS / MPG has lower antioxidant activity than 2% HBCS / MPG, indicating that HBCS / MPG hydrogel has good antioxidant properties, and HBCS may affect the antioxidant activity of melanin. Hydroxybutyl chitosan is a chitosan modified with hydroxyl groups, containing abundant amino and hydroxyl groups. Melanin is a complex polymer formed from tyrosine through multiple reactions, containing phenolic hydroxyl groups, etc. The amino groups of hydroxybutyl chitosan can bind to the phenolic hydroxyl groups of melanin through hydrogen bonds, electrostatic interactions, etc., altering the conformation of melanin molecules, affecting the exposure of its active sites and chemical reactivity. The hydroxybutyl chitosan molecular chains form steric hindrance around melanin, preventing external free radicals from contacting melanin and reducing its antioxidant effect.
[0070] Example 3: This embodiment verifies the effect of HBCS / MPG composite hydrogels with different concentrations (volume ratio of 7:3) through cell experiments.
[0071] 1. Assay of cell viability of hydrogels
[0072] Appropriate amounts of L929 cells and human nucleus pulposus cell suspensions were seeded into 48-well plates and cultured for 24 h. Then, 200 μL of HBCS2 / MPG2, HBCS2 / MPG3, HBCS2 / MPG4, HBCS3 / MPG3, and HBCS3 / MPG4 hydrogel solutions were added to each well, forming five experimental groups. The control group received 200 μL of physiological saline. After incubation for 24, 48, and 72 h, cell viability / death staining working solution was added, and staining was performed at 37°C in the dark for 20 min. The staining was then observed under a fluorescence microscope.
[0073] Two experimental groups were formed by adding 200 μL of HBCS3 / MPG4 and HBCS3 / MPG4+ADSCs-Exo (10 mM) hydrogel solutions to human nucleus pulposus cell suspensions, respectively. The control group was treated with 200 μL of physiological saline. After incubation for 24, 48, and 72 h, cell viability and death staining working solution was added, and staining was performed at 37°C in the dark for 20 min. The staining was then observed under a fluorescence microscope.
[0074] 2. Three-dimensional migration
[0075] L929 cells and human nucleus pulposus cells in good growth condition were centrifuged, resuspended, and diluted to 10 × 10⁻⁶. 4 Cells were counted at 1000 μL. 200 μL of cell suspension was added to the upper layer of the chamber, and HBCS2 / MPG2, HBCS2 / MPG3, HBCS2 / MPG4, HBCS3 / MPG3, and HBCS3 / MPG4 hydrogel solutions were added to the lower chamber as 5 experimental groups. 200 μL of physiological saline was added to the control group. The cells were cultured for 24 h, fixed with paraformaldehyde, stained with crystal violet, rinsed with distilled water, observed under a microscope, and cell counting analysis was performed.
[0076] Add 200 μL of HBCS3 / MPG4 and HBCS3 / MPG4+Exo hydrogel solutions to the human nucleus pulposus cell suspension as two experimental groups. Add 200 μL of physiological saline to the control group. Culture for 24 h, fix with paraformaldehyde, stain with crystal violet, rinse with distilled water, observe under a microscope, and perform cell counting analysis.
[0077] 3. Determination of reactive oxygen species scavenging capacity
[0078] Establishment of a human nucleus pulposus cell oxidative damage model
[0079] Human nucleus pulposus cells in the logarithmic growth phase were digested and centrifuged, then resuspended in DMEM medium containing 10% FBS and diluted to 2×10⁻⁶. 4200 μL of the culture medium was inoculated into 96-well plates (4000 cells per well) and cultured at 37°C for 24 h. After cell attachment, the old culture medium was discarded, and 200 μL of DMEM medium containing different concentrations of H2O2 (0, 10, 25, 50, 100, 200, 400, 600, and 800 μM) was added. The cells were then cultured at 37°C for 2 h to stimulate the production of reactive oxygen species. After the culture was completed, 20 μL of MTT assay solution was added, and the cells were incubated at 37°C in the dark for 4 h. Cell viability was then measured using a microplate reader.
[0080] The ROS scavenging ability of the hydrogel was detected using DCFH-DA fluorescence staining. Human nucleus pulposus cells at a concentration of 2 × 10⁻⁶ were used. 4 500 μL of cells / mL was seeded into 48-well plates, and 500 μL of serum-free DMEM medium containing 500 μM H2O2 was added. Then, sterile equal volumes of HBCS3 / MPG4 and HBCS3 / MPG4+Exo were added to the wells. The control group was replaced with fresh serum-free DMEM medium without H2O2, while the negative control group was only added with serum-free DMEM medium with a H2O2 concentration of 500 μM. After incubation at 37°C for 2 h, DCFH-DA was added, and the cells were incubated in the dark for 20 min. The intracellular fluorescence was observed and photographed using a fluorescence microscope.
[0081] To evaluate the protective effect of hydrogel in scavenging ROS on cells, human nucleus pulposus cells in the logarithmic growth phase were digested, centrifuged, resuspended in DMEM medium containing 10% FBS, and diluted to 2 × 10⁻⁶. 4 200 μL of the culture medium was inoculated into 96-well plates (4000 cells per well) and cultured at 37°C for 24 h. After cell attachment, the old culture medium was discarded, and 200 μL of 500 μM H2O2 was added to different concentrations of HBCS3 / MPG4 (25%, 50%, 75%, 100%) and HBCS3 / MPG4+Exo (25%, 50%, 75%, 100%). The control group was replaced with fresh serum-free DMEM medium without H2O2, while the negative control group was only inoculated with 500 μM H2O2 serum-free DMEM medium. After culturing at 37°C for 2 hours, 20 μL of MTT assay solution was added, and the cells were incubated at 37°C in the dark for 4 h. Cell viability was then measured using a microplate reader.
[0082] 4. Analysis of the in vitro anti-inflammatory effects of hydrogels
[0083] Logarithmic growth phase human nucleus pulposus cells were digested, centrifuged, and diluted to 10 × 10⁻⁶ using DMEM complete medium. 4Cells were seeded at a concentration of 100 μL / mL in 96-well plates (200 μL / well) to ensure adequate cell adhesion. The old culture medium was discarded, and 200 μL of HBCS3 / MPG4 solution and HBCS3 / MPG4+Exo solution were added to the experimental groups, respectively. The control group was cultured in serum-free DMEM medium. After culturing at 37°C for 2 hours, the cell culture supernatant was collected and centrifuged at 4000 rpm for 20 minutes to remove cell particles and polymers. The supernatant was stored at -20°C for later use. Working solutions for each component were prepared according to the kit instructions. Standard wells, zero-value wells, blank wells, and sample wells were set up in the 96-well plate. A standard detection system was established: gradient concentrations of standard solutions, sample diluents, and test samples were added to the reaction plate, with the blank wells kept dry. Enzyme-labeled antibody solution was added to the test wells, and the plate was sealed. Incubation was performed at 37°C in the dark to complete antigen-antibody binding. Multiple rinses with buffer were used to remove unbound material, and the wells were thoroughly dried after each rinse. The chromogenic substrate was mixed in the specified ratio and injected into each well. A second incubation in the dark was performed to observe the reaction signal. A stop solution was added to stabilize the chromogenic state, and the absorbance of each well was read at 450 nm using a spectrophotometer. The target protein content was calculated using a standard curve. These steps allow for accurate determination of IL-6 levels, providing crucial data for assessing the effects of hydrogels on human nucleus pulposus cells.
[0084] Results analysis: 1. By Figure 5 As shown in (Ⅰ), after 24 h of culture, the cells in the hydrogel group and the control group had normal morphology and were almost all green fluorescent. After 48 and 72 h of culture, the number of cells increased, but the morphology remained unchanged. Most of the cells were still green fluorescent, with no obvious red fluorescence. There was no significant difference between the L929 cell experimental group and the control group. The number of human nucleus pulposus cells was slightly higher in the experimental group than in the control group, indicating that HBCS / MPG hydrogel has the ability to promote cell growth. Figure 5 (II) When exosomes were added to the HBCS3 / MPG4 hydrogel solution and cultured for the same amount of time, the cells were stained again with Calcein-AM and PI dyes. It was observed that the cells cultured in the hydrogel solution with added exosomes grew slightly faster than the control group and the single hydrogel group, which once again proved that the hydrogel was non-toxic to cells and that exosomes had the ability to promote cell growth.
[0085] 2. For example Figure 6As shown in (I), Figures (A) and (B) depict the three-dimensional migration and number of L929 cells after 24 hours, while Figures (C) and (D) depict the three-dimensional migration and number of human nucleus pulposus cells after 24 hours. After 24 hours of migration, except for the HBCS2 / MPG4 group, the number of migrating cells in other groups was greater than that in the control group. Cell migration counting revealed that the hydrogels in each group either promoted or did not hinder the three-dimensional migration of L929 cells. HBCS2 / MPG2 and HBCS3 / MPG4 showed significant promoting effects, while the promoting effect on human nucleus pulposus cells was not significant. This demonstrates that the hydrogel is harmless to cells while providing a certain degree of migration promotion.
[0086] Figure 6 (II) The three-dimensional migration (A) and number of human nucleus pulposus cells after co-culturing with HBCS3 / MPG4 hydrogel and HBCS3 / MPG4+Exo hydrogel for 24 h. As shown in the figure, by culturing cells with exosome-loaded hydrogel medium and comparing them with the control group and cells cultured in medium with only hydrogel, it can be seen that the number of cells with exosomes added migrates more in three dimensions, and the promoting effect is stronger.
[0087] 3. From Figure 7(I) Observations revealed that in the control group, there was almost no green fluorescence signal, indicating that the production of reactive oxygen species (ROS) in cells was minimal under normal conditions, and the cell morphology remained normal. In contrast, in the control group with only H2O2 added, the cell morphology changed significantly, and strong green fluorescence appeared in the cells. This indicates that H2O2 caused the cells to produce a large amount of ROS, and the cells shrank, indicating that H2O2 caused oxidative stress damage to the cells. However, in the experimental groups with HBCS3 / MPG4 and HBCS3 / MPG4+Exo hydrogels, the green fluorescence signal was significantly weakened compared with the control group with only H2O2 added, indicating that the hydrogel can effectively inhibit the excessive production of ROS induced by H2O2. Further observation revealed that most cells in these experimental groups remained in good condition, with normal spindle-shaped cell morphology and no obvious signs of damage. This confirms that the hydrogel can effectively intervene in the oxidative stimulation of cells by H2O2, regulate the intracellular ROS level, maintain the dynamic balance between oxidation and antioxidation, thereby preventing the occurrence of oxidative stress response and playing a significant protective role for cells. As shown in Figure (II) A, H2O2 has no significant effect on cells at low concentrations. However, as the concentration increases, H2O2 leads to cell apoptosis. The figure also shows that H2O2 concentrations below 400 μM have little effect on L929 cells, consistent with the trend in the control group. However, when the H2O2 concentration is above 400 μM, cell viability is below 20%. Therefore, H2O2 concentrations between 400 μM and 600 μM have a significant impact on cell viability. Thus, a H2O2 concentration of 500 μM was chosen to study the protective effect of hydrogels on cells.
[0088] As shown in Figure (II) B, the figure illustrates the effects of different concentrations of HBCS3 / MPG4 and HBCS3 / MPG4+Exo on cell growth. With HBCS3 / MPG4 alone, cell growth was fastest at 25 μM, approximately 100%. Subsequently, as the concentration increased to 50 μM, 75 μM, and 100 μM, the growth rate decreased slightly but remained above 80%. In the HBCS3 / MPG4+Exo group, cell migration was significantly increased at all concentrations, especially at 75 μM and 100 μM, where the migration rate approached 120%. This indicates that the addition of exosomes significantly enhanced the cell migration-promoting effect of the hydrogel.
[0089] 4. IL-6 (interleukin-6) is an important inflammatory cytokine, and its expression level can reflect the inflammatory state of cells. Hydrogels can exert anti-inflammatory effects by regulating IL-6 expression in cells. The anti-inflammatory properties of hydrogels can be evaluated by measuring IL-6 expression in cells after hydrogel treatment. Experiments showed that the control group had the lowest IL-6 cytokine level, approximately 100 pg / mL; the HBCS3 / MPG4 group showed a significantly increased IL-6 cytokine level, approximately 150 pg / mL; and the HBCS3 / MPG4+Exo group had the highest IL-6 level, approaching 400 pg / mL. This indicates that the HBCS3 / MPG4 hydrogel can effectively promote IL-6 expression, and the addition of exosomes further significantly increased the IL-6 cytokine expression level, suggesting that exosomes enhance the expression of the anti-inflammatory cytokine IL-6 in the hydrogel system.
[0090] Based on the analysis of the above-mentioned hydrogel properties, the concentration of the HBCS / MPG composite hydrogel selected in Example 4 is 3% HBCS and 4% MPG, with a volume ratio of 7:3.
[0091] Example 4:
[0092] Preparation of the experiment: SD rats (weighing 200-250 g) were selected and acclimatized for one week. Before surgery, the fur on the back of the rats was shaved, and the surgical area was disinfected with iodine and alcohol. The rats were anesthetized by intraperitoneal injection of 3% (w / v) sodium pentobarbital solution (1 mL / kg). The anesthetized rats were fixed prone on the operating table, ensuring that the caudal vertebrae were exposed. The position of the caudal vertebrae was observed by X-ray, and the specific locations of the experimental caudal vertebrae Co5-6 and Co7-8 were determined by using a syringe needle. A 22-gauge injection needle was inserted perpendicularly to the intervertebral disc, gently rotated and advanced, ensuring that the needle passed through the annulus fibrosus and entered the nucleus pulposus. The needle was rotated 180° and held in position for 30 seconds, then slowly withdrawn. After the model was successfully established, the control group was injected with 20 μL of physiological saline into the Co5-6 vertebral segment, and the experimental group and the experimental group of HBCS / MPG composite hydrogel were injected with 20 μL of HBCS / MPG4+ ADSCs-Exo (10mM) into the Co7-8 vertebral segment. Each group was equipped with 3-5 SD rats with the same growth status, and the control group and the two experimental groups used the same SD rat but different vertebral segments.
[0093] Animal experiments:
[0094] 1. Degradability and biocompatibility testing of hydrogels
[0095] SD rats weighing 200-220 g were selected and fasted for 12 hours before the experiment, but with free access to water. Irradiation-sterilized HBCS3 / MPG4 hydrogel lyophilized product was prepared into a 2% (w / v) solution with physiological saline. Rats were anesthetized by intraperitoneal injection of a 3% (w / v) sodium pentobarbital solution (1 mL / kg).
[0096] In the subcutaneous degradation experiment, after shaving the back, 0.1 mL of hydrogel solution was injected subcutaneously using a 1 mL syringe; in the muscle degradation experiment, after shaving the legs, the skin of the legs was cut open with a scalpel, and 0.1 mL of hydrogel solution was injected under the first layer of muscle using a 1 mL syringe, and the wound was sutured (the same SD rat was used for both the subcutaneous degradation and muscle degradation experiments).
[0097] 1 mL of penicillin solution (40,000 units) was injected to prevent postoperative infection. Rats were euthanized by cervical dislocation at 1, 2, 4, and 6 weeks. The skin and muscle around the injection site were cut open to observe the degradation of the hydrogel and the gross morphology of the surrounding tissues. The hydrogel and surrounding tissue were excised, fixed in formaldehyde, and rinsed overnight. Then, tissue blocks from the back and legs were trimmed, graded for dehydration, paraffin embedding, and sectioned. The inflammatory response was observed by H&E staining. Microscopic observation and photography were used to assess the hydrogel degradation. The degree of inflammatory response was assessed using H&E staining results.
[0098] 2. Blood compatibility test of hydrogels
[0099] To assess the blood compatibility of the hydrogel material, an observational experiment was conducted observing changes in hematological parameters. Fresh rabbit blood was collected and processed to ensure safety upon contact with the hydrogel. The specific steps were as follows: Fresh rabbit blood was collected using a sodium citrate negative pressure blood collection tube, mixed with an equal volume of physiological saline, and placed in a 15 mL centrifuge tube. The mixture was centrifuged at 1500 r / min at 4℃ for 10 min, the supernatant was removed, and the centrifugation was repeated 4-6 times until the supernatant was clear. Red blood cells from the bottom of the tube were collected and prepared into a 2% (v / v) suspension with physiological saline, which was then stored at 4℃ for later use.
[0100] The irradiated sterilized HBCS3 / MPG4 hydrogel lyophilized product was prepared into solutions of different concentrations (2 mg / mL, 1 mg / mL, and 500 μg / mL) with physiological saline. An equal volume of this solution was mixed with a red blood cell suspension. Physiological saline and distilled water were used as negative and positive controls, respectively. The mixture was incubated at 37°C for 1 h. After incubation, the supernatant was collected by centrifugation, and the absorbance at 545 nm was measured. The experiment was conducted in parallel groups to ensure data reliability, and the hemolysis rate was calculated.
[0101] 3. Rat caudal vertebral magnetic resonance imaging
[0102] Rats were anesthetized with sodium pentobarbital at 1, 2, and 4 weeks after the injury modeling experiment. Their tails were then marked, and T1-weighted imaging (T1WI) and T2-weighted imaging (T2WI) sequences were used to assess the water content and structural changes of the intervertebral discs. Signal intensity on T2WI was observed; high signal indicated high water content, and low signal indicated water loss. The experimental groups were injected with HBCS3 / MPG4 and HBCS3 / MPG4+Exo, while the control group was injected with saline.
[0103] 4. HE staining observation of nucleus pulposus tissue
[0104] On days 7, 14, and 28 after treatment in both the experimental and control groups, tissues were fixed and rinsed with water for 12 hours. The tissue blocks were then trimmed into regular shapes and dehydrated sequentially for 2 hours each with 50%, 70%, and 95% ethanol and anhydrous ethanol I and II. After dehydration, the tissues were soaked in xylene for 15 minutes, followed by 15 minutes in fresh xylene to achieve transparency. Finally, the tissue blocks were embedded after soaking in paraffin I, II, and III for 1 hour each. After standardized embedding, the samples were fixed in a sectioning apparatus, thin sections were prepared, and transferred to a temperature-controlled water bath for tissue expansion. Anti-detachment slides were used for sample positioning, and the samples were dried and cured under constant temperature conditions. Gradient dewaxing was then performed: lipid components were removed sequentially through a xylene system and mixed solvents, followed by stepwise rehydration through a gradient of ethanol concentrations. In the two-color staining system, a basic staining agent was first used to mark nuclear structures, followed by an acidic differentiation agent to optimize the staining effect. Then, an acidic staining agent was used to perform composite staining of cytoplasmic components. After staining, the samples were dehydrated using a reverse ethanol gradient, the liquid medium was replaced with a clearing agent, and finally the sample structure was sealed with neutral resin. Pathological features were then obtained using an optical microscope imaging system. Two experimental groups were selected: one injected with HBCS3 / MPG4 and the other with HBCS3 / MPG4+Exo. The saline injection group served as the control group, and the group without the injury model treatment was the healthy group.
[0105] 5. Masson staining observation of nucleus pulposus tissue
[0106] The tissue sections described above were subjected to Masson staining according to the kit instructions. First, the tissue sections were dewaxed to water, and nuclear staining was performed using hematoxylin. Following differentiation with acidic ethanol and rinsing with running water, the sections were stained in Masson staining solution, resulting in a blue color for the collagen fibers. Differentiation was then performed using phosphotungstic acid solution to enhance staining contrast. The sections were counterstained with hematoxylin counterstaining solution, resulting in a red color for the cell nuclei. Finally, after dehydration and clearing, the sections were mounted with neutral resin, air-dried, and observed under a microscope to assess the collagen fiber condition of each wound group. Two experimental groups were selected: one injected with HBCS3 / MPG4 and the other with HBCS3 / MPG4+Exo. The saline injection group served as the control group, and the group without injury model treatment was the healthy group.
[0107] 6. Immunofluorescence staining of nucleus pulposus tissue
[0108] Immunofluorescence staining, based on the specific binding of antigen and antibody, can visually display the distribution and expression of IL-6 and TNF-α in nucleus pulposus tissue. In the experiment, nucleus pulposus tissue was first obtained, fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into 5μm sections. After dewaxing and hydration, the antigen was repaired with citrate buffer, and non-specific binding was blocked with normal goat serum. Diluted anti-IL-6 primary antibody was added, and the sections were incubated at 37°C for 2 hours. Then, fluorescently labeled secondary antibody was added, and the sections were incubated at 37°C in the dark for 1 hour. Finally, the cell nuclei were stained with DAPI, and the sections were mounted with an anti-fluorescence quencher. The sections were observed and photographed under a fluorescence microscope, and the location and expression were determined based on the fluorescence signal. This method has high specificity and sensitivity, providing a visual basis for studying the inflammation and pathological mechanisms of intervertebral discs.
[0109] Results analysis:
[0110] 1. The biodegradability and biocompatibility of the hydrogel in vivo were observed by subcutaneous and intramuscular injection into rats. Figure 8 As shown in (I), the subcutaneous hydrogel gradually decreased in volume and lightened in color over time. By week 8, the hydrogel had essentially degraded, leaving only some redness under the skin. The hydrogel in the muscle degraded more slowly than the subcutaneous hydrogel. In weeks 1 and 2, its volume only decreased slightly, but it also gradually degraded over time. HE staining also showed that in the first week after the hydrogel entered the body, inflammatory reactions occurred in both the subcutaneous tissue and muscle. As the hydrogel is a foreign implant, rejection is a normal phenomenon. By week 4, the inflammatory reaction had significantly decreased, and there was no damage to the skin and muscle. This indicates that the hydrogel can stably degrade in both the subcutaneous tissue and muscle without harming the body.
[0111] Depend on Figure 8(II) It can be seen that in the first week after hydrogel implantation, the hydrogel is easily observable due to its melanin content. At this time, the hydrogel is still relatively large and adheres tightly to the skin and muscles. Different degrees of inflammatory reactions occur in both the dermis and muscles. The more severely inflamed areas are dark purplish-red in color, with a large number of inflammatory cells produced, showing obvious changes. In the second week after hydrogel implantation, it can be seen that the volume of the hydrogel is decreasing, and the skin tissue is slowly recovering. The inflammatory reaction has lessened, and the implanted area is light purplish-red, with a range similar to that of the first week, indicating that the inflammation has not spread. In the fourth week after hydrogel implantation, the hydrogel has obviously partially degraded, with its volume only 50% of its original size. The black pigment cannot penetrate the skin, and the color is not obvious. According to the staining results, the inflammatory reaction has been greatly reduced, and the implanted area is dark pink, indicating that the inflammatory reaction has basically stopped. In the eighth week after hydrogel implantation, the hydrogel has completely degraded, and no remaining part can be found, indicating that its degradation is good, the inflammatory reaction has completely disappeared, the implanted area is light pink, and both the subcutaneous tissue and muscles have recovered.
[0112] 2. Figure 8 (III) This study demonstrates the hemolytic phenomenon after contact between hydrogels of different concentrations and blood, and further quantifies the hemolysis rate of hydrogels at different concentrations. From left to right, the hydrogels are H2O (positive control), NaCl (negative control), 2 mg / mL hydrogel, 1 mg / mL hydrogel, and 0.5 mg / mL hydrogel. It can be observed that the hemolysis phenomenon gradually decreases with decreasing hydrogel concentration, indicating a positive correlation between the hemolysis rate and concentration. The results show that the 2 mg / mL hydrogel has the highest hemolysis rate, approximately 0.4%, the 1 mg / mL hydrogel has a hemolysis rate of approximately 0.3%, and the 0.5 mg / mL hydrogel has the lowest hemolysis rate, approximately 0.08%. This indicates that the hydrogel has better blood compatibility at lower concentrations and less damaging effect on erythrocytes. Overall, the hemolysis rate of the hydrogel decreases significantly with decreasing concentration, showing a good dose-dependent effect.
[0113] 3. Four weeks after treatment, an MRI scan of the intervertebral disc was performed. Figure 9 The results of MRI (A) and water content (B) of the intervertebral disc are presented by... Figure 9 It was observed that after successful modeling, compared to the untreated vertebral segments, the signal intensity of the intervertebral discs in the acupuncture-treated group was weakened, and they lost moisture, appearing dark black, indicating successful modeling. One week later, the signal intensity of the intervertebral discs in the treated group increased, and the moisture content increased, turning them light black. With further time, the moisture content within the intervertebral discs increased, and the color gradually changed from black to bright white, signifying gradual disc recovery. The HBCS3 / MPG4 group showed significantly higher brightness than the control group at 1, 2, and 4 weeks, indicating substantial growth. Figure 9The quantitative results of (B) also show this trend; and the HBCS3 / MPG4+Exo group showed significantly better signal intensity than the HBCS3 / MPG4 group and the control group at 2 weeks, and the signal intensity reached its strongest at 4 weeks, indicating that the water was restored. Therefore, it can be concluded that the HBCS3 / MPG4+Exo group has a significant repair effect on the degenerated nucleus pulposus.
[0114] 4. The recovery of the intervertebral disc was observed by HE staining of tissue sections. Figure 10 The tissue responses of different treatment groups at 0 weeks (0w), 2 weeks (2w), and 4 weeks (4w) were observed. The healthy group showed stable tissue structure and no significant pathological changes at all time points, providing a benchmark for evaluating the effects of other treatment groups. In the control group, tissue structure gradually changed over time, with increased inflammation and fibrosis, which may reflect the natural pathological process of intervertebral disc degeneration. In the HBCS3 / MPG4 group, the purple area of the nucleus pulposus deepened at 2 weeks, indicating significant changes in tissue structure. By 4 weeks, the nucleus pulposus had shown significant repair, reduced inflammation, and less fibrosis than the control group, with some recovery effect, suggesting that hydrogel treatment may have a protective effect. At all time points, the HBCS3 / MPG4+Exo group showed the least changes in tissue structure compared to the HBCS3 / MPG4 group and the control group. At 2 weeks, the purple color of the nucleus pulposus decreased and the inflammatory response declined. By 4 weeks, the degree of fibrosis was the lowest compared to other groups. This suggests that hydrogel plus exosome treatment may have significant advantages in promoting intervertebral disc tissue repair and reducing inflammatory response.
[0115] 5. Through observation Figure 11 Masson staining sections were used to analyze the collagen fiber content and potential degree of fibrosis in intervertebral disc tissue under different treatment conditions. In the healthy control group, the blue area was relatively small, indicating moderate collagen fiber content and normal tissue structure. Over time, the blue area in the control group gradually increased, especially at week 4, which may indicate collagen fiber proliferation and suggest an increased degree of fibrosis. In contrast, the HBCS3 / MPG4 group showed an increase in blue area at weeks 2 and 4, but to a lesser extent compared to the control group, which may indicate that hydrogel treatment has a certain inhibitory effect on collagen fiber proliferation. The most significant effect was observed in the HBCS3 / MPG4+Exo group, where the blue area was significantly less than in other groups at all time points, especially at week 4, indicating that this treatment significantly reduced collagen fiber proliferation and may have a positive effect on alleviating fibrosis. The staining results suggest that hydrogel plus exosome treatment may have a significant advantage in inhibiting collagen fiber proliferation and alleviating intervertebral disc tissue fibrosis, providing a promising strategy for the treatment of intervertebral disc degeneration.
[0116] 6. Immunofluorescence is based on the principle of antigen-antibody specific binding, constructing a signal labeling system. In the experiment, a fluorescently conjugated secondary antibody forms a complex with the primary antibody bound to the target antigen, and emission spectra at characteristic wavelengths are generated by laser excitation. For example... Figure 12 The results showed that in the second week, all three groups exhibited fluorescence, but the fluorescence intensity of the control group and the HBCS3 / MPG4 group was higher than that of the HBCS3 / MPG4+Exo group. In the fourth week, the fluorescence intensity of the control group further increased, while the fluorescence intensity of both the HBCS3 / MPG4 group and the HBCS3 / MPG4+Exo group decreased, with the HBCS3 / MPG4+Exo group showing a lower intensity than the HBCS3 / MPG4 group. This indicates that the HBCS3 / MPG4+Exo hydrogel can effectively inhibit IL-6 expression and reduce the inflammatory response, with better results than the hydrogel alone. This phenomenon may be related to the abundant bioactive molecules in exosomes, which can regulate inflammation-related signaling pathways and inhibit the production and release of pro-inflammatory factors, thereby more effectively reducing the inflammatory response.
[0117] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. An injectable temperature-sensitive hydrogel, characterized in that, The hydrogel is composed of hydroxybutyl chitosan (HBCS) and melanin-polyethylene glycol / gelatin core-shell microspheres (MPG).
2. The injectable thermosensitive hydrogel as described in claim 1, characterized in that, The mass ratio of HBCS to MPG is 1-27:1-4; the hydrogel has a porous network structure with uniform porosity distribution, a compact structure, and small pore size; the storage modulus G' is higher than the loss modulus G''; and the swelling ratio is high. It exhibits good stability, and the hydrogel remains stable at body temperature (37°C); the HBCS / MPG hydrogel also possesses excellent antioxidant properties.
3. The method for preparing the injectable thermosensitive hydrogel according to claim 1, characterized in that, Includes the following steps: (1) Preparation of hydroxybutyl chitosan HBCS; (2) Preparation of melanin-polyethylene glycol / gelatin core-shell microspheres (MPG); (3) Dissolve the prepared HBCS and MPG in deionized water, stir thoroughly, and then mix the HBCS solution and MPG solution. The mass ratio of HBCS and MPG is 1-27:1-4. The mixture is ready to form a gel.
4. The preparation method according to claim 3, characterized in that, The specific steps (1) are as follows: chitosan is added to distilled water and stirred until mixed. Acetic acid is added and then NaOH is slowly added dropwise until alkaline. The mixture is stirred until a latex-like precipitate is obtained. The white precipitate is added to isopropanol and water and mixed and stirred. 1,2-epoxybutane is added dropwise to continue the reaction. After the reaction is completed, the mixture is cooled to room temperature. The mixture is dialyzed, centrifuged, and the supernatant is freeze-dried to obtain HBCS.
5. The preparation method according to claim 3, characterized in that, Step (2) includes: 1) Dissolve NH2-PEG-NH2 and melanin separately in Tris buffer and stir until completely dissolved; then weigh EDC and NHS and add them to the above solution, followed by PEG solution and react for one day; after the reaction, remove impurities with anhydrous ethanol, wash the precipitate three times, dialyze and freeze dry to obtain MP product; 2) Weigh gelatin and add it to MES buffer for hydration until the solution is clear and transparent; then add EDC and NHS to the solution and stir at room temperature until completely dissolved; weigh MP and dissolve it in Tris solution and stir overnight until dissolved; mix the two reaction solutions, react at room temperature, dialyze, and freeze dry to obtain MPG.
6. The preparation method according to claim 3, characterized in that, In step (3), the prepared HBCS is dissolved in deionized water to prepare solutions with concentrations of 2% and 3% (w / v), and the prepared MPG is dissolved in deionized water to prepare solutions with concentrations of 2%, 3%, and 4% (w / v). After thorough mixing, the HBCS solutions and MPG solutions of different concentrations are mixed in different proportions until a gel is formed.
7. The use of the injectable thermosensitive hydrogel according to claim 1 in the preparation of antioxidant and anti-inflammatory products.
8. The use of the injectable thermosensitive hydrogel according to claim 1 in the preparation of intervertebral disc degeneration nucleus pulposus repair products.
9. The application of the injectable thermosensitive hydrogel mixed with exosomes (EXO) as described in claim 1 in the preparation of antioxidant and anti-inflammatory products.
10. The use of the injectable thermosensitive hydrogel mixed with exosomes (EXO) as described in claim 1 in the preparation of intervertebral disc degeneration nucleus pulposus repair products.