Preparation method of chitosan-mussel mucin bionic polypeptide-SVF composite hydrogel
By preparing a chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel, the problems of short residence time and insufficient adhesion of SVF in the joint cavity were solved, achieving long-term fixation of SVF and improving the therapeutic effect.
Patent Information
- Application Number
- CN202511760440.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-27
- Publication Date
- 2026-01-23
AI Technical Summary
In existing technologies, SVF injected into the joint cavity alone has a short residence time and is easily removed. Hyaluronic acid cannot effectively fix SVF, and chitosan hydrogel has insufficient adhesion to cartilage tissue, affecting the therapeutic effect.
A chitosan-mussel adhesive protein biomimetic peptide-SVF composite hydrogel was prepared by mixing mussel adhesive protein biomimetic peptides and thiolized chitosan to form a hydrogel precursor solution, which was then gently mixed with SVF to form a composite hydrogel with strong adhesion and good biocompatibility.
Hydrogels can adhere firmly to the cartilage surface, prolong the residence time of SVF, promote cell colonization and function, improve treatment efficacy, and have good biocompatibility and injectability.
Smart Images
Figure CN121371307A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomaterials technology, and in particular to a method for preparing and applying a chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel. Background Technology
[0002] Osteoarthritis (OA) is a common chronic degenerative joint disease characterized by progressive destruction of articular cartilage, synovitis, subchondral bone remodeling, and osteophyte formation, leading to joint pain, stiffness, and functional impairment. Current clinical treatments mainly include drug therapy, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and analgesics; physical therapy; and surgical treatment, such as joint replacement surgery. However, these methods are mostly symptomatic treatments and are unlikely to reverse cartilage damage.
[0003] In recent years, cell-based therapeutic strategies, particularly the application of adipose-derived stromal vascular fraction (SVF), have offered new prospects for the treatment of osteoarthritis (OA). SVF is rich in various active cell types, including mesenchymal stem cells (MSCs), endothelial progenitor cells, and immune cells, and possesses multiple functions such as anti-inflammatory effects, immunomodulation, and promotion of tissue repair and regeneration. Clinical studies have shown that intra-articular injection of SVF can alleviate pain and improve joint function in OA patients.
[0004] SVF therapy utilizes the synergistic effects of multiple cells within SVF to exert anti-inflammatory, immunomodulatory, and tissue repair functions. Hydrogels are polymeric materials with a three-dimensional network structure that can mimic the extracellular matrix, providing a supportive and growth-promoting microenvironment for cells, while also exhibiting good biocompatibility and injectability. Chitosan is a natural polysaccharide with good biocompatibility, biodegradability, antibacterial properties, and wound-healing effects. Mussel adhesive protein biomimetic peptides mimic the strong underwater adhesion of mussels. By synthesizing peptides with specific amino acid sequences (such as those containing DOPA and RGD sequences), the material achieves a firm bond with tissue and promotes cell adhesion.
[0005] SVF intra-articular injection alone: Freshly isolated SVF is injected directly into the patient's joint cavity. SVF combined with hyaluronic acid: SVF is mixed with hyaluronic acid and injected into the joint cavity; the hyaluronic acid acts as a lubricant and temporary carrier. Chitosan hydrogel: Chitosan hydrogel can be used as a carrier for drugs or cells in tissue engineering and regenerative medicine.
[0006] The existing technology has the following drawbacks: SVF has a short residence time in the joint cavity after injection and is easily removed, affecting the efficacy; although hyaluronic acid has a lubricating effect, it cannot effectively fix SVF and degrades relatively quickly; although chitosan hydrogel can be used as a carrier, it lacks strong adhesion to cartilage tissue, and the microenvironment of chitosan hydrogel alone is not conducive to the long-term survival and function of cells in SVF. Summary of the Invention
[0007] The technical problem to be solved by the present invention is to provide a method for preparing and applying a chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel, which addresses the shortcomings of the prior art. The aim is to provide a composite hydrogel with good biocompatibility, strong tissue adhesion, injectability, and the ability to promote SVF cell colonization, survival, and function, for intra-articular injection therapy of osteoarthritis, so as to improve the efficacy of SVF therapy.
[0008] This invention provides a method for preparing a chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel, comprising the following steps:
[0009] S1. Preparation of mussel adhesive protein biomimetic peptides: The Fmoc solid-phase peptide synthesis method was adopted, using Wang resin and Fmoc-protected amino acids as monomers. The peptide chains of the open peptides were gradually extended through deprotection, condensation, and washing. The peptides were cut and purified to obtain purified peptides. The purified peptides were dissolved in buffer solution for modification. After the reaction was completed, the peptides were purified again and freeze-dried to obtain mussel adhesive protein biomimetic peptides.
[0010] S2. Preparation of thiolated chitosan: Chitosan was dissolved in acetic acid solution to obtain a mixed solution. Triton B was added to the mixed solution and the mixture was stirred at room temperature. After the reaction was completed, the pH value of the solution was adjusted, and then dialyzed in deionized water using a dialysis bag. The dialyzed solution was freeze-dried to obtain thiolated chitosan.
[0011] S3. Preparation of SVF: Fat aspiration was performed using standard tumescent anesthesia. The aspirated adipose tissue was washed with PBS to remove blood cells and free lipid droplets. Then, type I collagenase solution was added, and the mixture was shaken in a water bath for enzymatic digestion. After digestion, the supernatant was removed by centrifugation once to remove the upper lipid droplets and undigested tissue. The supernatant was discarded, and the cell pellet was resuspended in PBS. The cell suspension was filtered through a cell sieve to remove tissue debris. The cell pellet was centrifuged a second time to remove the supernatant, and the cell pellet was resuspended in PBS. After washing, the number of SVF cells was counted using a hemocytometer, and the cell concentration was adjusted with DMEM / F12 cell culture medium to obtain SVF.
[0012] S4. Preparation of composite hydrogel:
[0013] S41. Preparation of mussel adhesive protein biomimetic polypeptide solution: Dissolve mussel adhesive protein biomimetic polypeptide in PBS to prepare mussel adhesive protein biomimetic polypeptide solution.
[0014] S42. Preparation of thiolated chitosan solution: Dissolve thiolated chitosan in acetic acid solution to prepare thiolated chitosan solution;
[0015] S43. Mixing of hydrogel precursor solution: Mix mussel adhesive protein biomimetic polypeptide solution and thiolized chitosan solution, gently vortex at room temperature to mix, and let stand to form hydrogel precursor solution.
[0016] Preparation of S44 and SVF-hydrogel composite system: SVF was gently mixed with the hydrogel precursor solution in S43 to obtain the final chitosan-mussel adhesive protein biomimetic peptide-SVF composite hydrogel.
[0017] According to the preparation method of chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel provided by the present invention, the condensing agent used in S1 is HBTU / HOBt, the condensation time is 2 h, the polypeptide cleavage solution is a TFA / TIS / H2O solution with a volume ratio of 95:2.5:2.5, the purification method is reversed-phase high-performance liquid chromatography, the purity of the purified polypeptide is >95%, the buffer is maleimide-NHS ester, the freeze-drying temperature is -35 ℃, and the freeze-drying time is 36 h.
[0018] According to the preparation method of chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel provided by the present invention, in step S2, the degree of deacetylation of chitosan is ≥90%, its molecular weight is 150 kDa, the concentration of the acetic acid solution is 0.5%–2%, the concentration of the mixed solution is 1%–3%, the molar ratio of Triton B to chitosan is 1:5–15, the stirring reaction time is 12–36 h, the solvent for adjusting the pH value of the solution is a 1 mol / L HCl solution, the target pH value of the pH adjustment solution is 2.0–6.0, the molecular weight cutoff of the dialysis bag is 3500 Da, the dialysis time is 36–54 h with deionized water replaced every 8 h, the freeze-drying temperature is -35 ℃, and the freeze-drying time is 36 h.
[0019] According to the preparation method of chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel provided by the present invention, the mass concentration of the type I collagenase solution in S3 is 0.075%, the temperature of the water bath is 37 ℃, the enzyme digestion time is 60 min, the centrifugal force of the first centrifugation is 1200 g, the centrifugation time is 10 min, the pore size of the cell sieve is 100 μm, the centrifugal force of the second centrifugation is 400 g, the centrifugation time is 5 min, and the cell concentration of the SVF is 1×10⁻⁶. 7 cells / mL.
[0020] According to the preparation method of chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel provided by the present invention, the ratio of mussel adhesive protein biomimetic polypeptide to PBS in S41 is 10 mg: 0.5 mL, the pH value of PBS is 7.4, and the concentration of mussel adhesive protein biomimetic polypeptide solution is 20 mg / mL.
[0021] According to the preparation method of chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel provided by the present invention, the ratio of the amount of thiolated chitosan to acetic acid solution in S42 is 10-30 mg: 0.5 mL, the concentration of the acetic acid solution is 0.5%-2%, and the concentration of the thiolated chitosan solution is 20-60 mg / mL.
[0022] According to the preparation method of chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel provided by the present invention, the ratio of the mussel adhesive protein biomimetic polypeptide solution and the thiolated chitosan solution in step S43 is 0.1-1 mL: 0.5 mL, and the standing time is 10-50 min.
[0023] In the preparation method of chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel provided by the present invention, the amount of SVF used in S44 is 1 mL.
[0024] The present invention also provides an application of the above-mentioned chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel, wherein the chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel is used to promote cartilage regeneration.
[0025] Compared with the prior art, the present invention has the following advantages:
[0026] This invention provides a method for preparing a chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel. The mussel adhesive protein biomimetic polypeptide endows the hydrogel with strong adhesive ability, enabling it to adhere firmly to the cartilage surface and prevent SVF loss. The hydrogel, as a carrier of SVF, can fix it within the joint cavity, prolonging its residence time and continuously exerting its therapeutic effect. Both chitosan and the mussel adhesive protein biomimetic polypeptide have good biocompatibility and will not cause immune rejection. The RGD sequence in the polypeptide can promote cell adhesion, and chitosan provides a favorable cell growth microenvironment, which is beneficial for cell colonization, survival, and function within the SVF. Chitosan itself has a certain cartilage repair-promoting effect, and its synergistic effect with SVF can more effectively promote cartilage regeneration. The hydrogel precursor solution has good fluidity and can be injected into the joint cavity via a minimally invasive procedure, making the operation simple. Attached Figure Description
[0027] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0028] Figure 1 A schematic diagram of the preparation process of chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel;
[0029] Figure 2 This is the mass spectrum of the MAP peptide;
[0030] Figure 3 This is a high-performance liquid chromatogram of the MAP peptide;
[0031] Figure 4 SEM morphology and EDS elemental distribution of C / N / O / S in SH-chitosan hydrogel;
[0032] Figure 5 SEM morphology and EDS plots of C / N / O / S elements of SH-chitosan-MAP hydrogel;
[0033] Figure 6 FTIR images of SH-chitosan hydrogel and SH-chitosan-MAP hydrogel;
[0034] Figure 7 XPS for SH-chitosan hydrogel;
[0035] Figure 8 XPS image of SH-chitosan-MAP hydrogel;
[0036] Figure 9Rheological diagrams of SH-chitosan hydrogel and SH-chitosan-MAP hydrogel;
[0037] Figure 10 Image showing the results of a Micro-CT scan of the knee joint;
[0038] Figure 11 Image of safranin-fixing green staining of the knee joint. Detailed Implementation
[0039] Example 1
[0040] This embodiment provides a chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel, which is composed of mussel adhesive protein biomimetic polypeptide, chitosan and SVF.
[0041] A schematic diagram of the preparation process of chitosan-mussel adhesive protein biomimetic peptide-SVF composite hydrogel is shown below. Figure 1 As shown;
[0042] The preparation method of chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel is as follows:
[0043] The preparation method of S1, mussel adhesive protein biomimetic polypeptide is as follows:
[0044] The sequence “GGGRGDSPPP**YDK(DOPA)YDK(DOPA)**K-NH2” was designed by modifying the ε-amino group of lysine (K) with a maleimide group, where DOPA is 3,4-dihydroxyphenylalanine and RGD is arginine R-glycine G-aspartic acid D sequence.
[0045] The Fmoc (fluorenemethyloxycarbonyl) solid-phase peptide synthesis method was adopted, using Wang resin (p-benzyl alcohol polystyrene resin) and Fmoc-protected amino acids as monomers. The peptide chain was gradually extended through deprotection, condensation, and washing steps. The condensation agent selected was HBTU / HOBt (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate / 1-hydroxybenzotriazole), and the condensation time was 2 hours.
[0046] After the peptide synthesis was completed, the peptide was cleaved using a TFA / TIS / H2O (volume ratio of 95:2.5:2.5, trifluoroacetic acid / triisopropylsilane / water) mixed solution and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain peptides with a purity greater than 95%.
[0047] Finally, the purified peptide was dissolved in a buffer containing maleimide-NHS ester (excess, N-hydroxysuccinimide ester group) for maleimide group modification. After the reaction was completed, it was purified again and freeze-dried at -35 ℃ for 36 h to obtain the final mussel adhesive protein biomimetic peptide (hereinafter referred to as MAP peptide).
[0048] The sequence design of MAP peptides, which includes DOPA and RGD sequences, is key to achieving adhesion and cell adhesion.
[0049] The mass spectrum of the MAP peptide is as follows: Figure 2 As shown, the mass spectrometry results are consistent with the theoretical molecular weight of 2783.00 Da, confirming that the synthesized peptide is the target product; the high-performance liquid chromatogram of the MAP peptide is shown below. Figure 3 As shown, the main peak accounts for 95.52% of the total area, indicating that the mussel polypeptide has a very high purity.
[0050] S2. Preparation of thiolized chitosan:
[0051] In this embodiment, the chitosan is purchased medical-grade chitosan (Sigma-Aldrich) with a degree of deacetylation ≥90% and a molecular weight of 150 kDa (viscosity-average molecular weight).
[0052] Chitosan was dissolved in a 1% (w / v) acetic acid solution to prepare a 2% (w / v) solution;
[0053] Add Triton B (Traut's Reagent, 2-Iminothiolane hydrochloride, commercially available from Sigma-Aldrich), with a molar ratio of Triton B to chitosan of 1:10, and stir the reaction at room temperature for 24 hours.
[0054] After the reaction was completed, the pH was adjusted to 4.0 using 1M HCl solution, and then dialyzed in deionized water for 48 h using a dialysis bag with a molecular weight cutoff of 3500 Da, with the deionized water being changed every 8 h.
[0055] Finally, the dialyzed solution was freeze-dried at -35 °C for 36 h to obtain thiolated chitosan (hereinafter referred to as SH-chitosan).
[0056] S3. Preparation of SVF (stromal vascular component):
[0057] In this embodiment, the source of SVF was selected as rat autologous adipose tissue, such as subcutaneous fat from the abdomen or thigh.
[0058] Under aseptic conditions, fat aspiration was performed using standard tumescent anesthesia. The aspirated adipose tissue was washed three times with PBS (Phosphate Buffered Saline) to remove blood cells and free lipid droplets. Then, 0.075% (w / v) type I collagenase solution was added, and the mixture was incubated at 37 °C with shaking for 60 min. After digestion, the mixture was centrifuged at 1200 g for 10 min to remove the upper lipid droplets and undigested tissue. The supernatant was discarded, and the cell pellet was resuspended in PBS. The cell suspension was filtered through a 100 μm cell sieve to remove tissue debris. The mixture was centrifuged at 400 g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in PBS, and the washing was repeated twice. The number of SVF cells was counted using a hemocytometer, and the cell concentration was adjusted to 1 × 10⁶ cells / mL using DMEM / F12 cell culture medium. 7 cells / mL, to obtain SVF;
[0059] S4. Preparation of composite hydrogel:
[0060] S41. Preparation of MAP peptide solution: Dissolve 10 mg of MAP peptide in 0.5 mL of PBS (pH 7.4) to prepare a MAP peptide solution with a concentration of 20 mg / mL.
[0061] S42, Preparation of SH-Chitosan Solution: Dissolve 20 mg of SH-chitosan in 0.5 mL of 1% (v / v) acetic acid solution to prepare a SH-chitosan solution with a concentration of 40 mg / mL, i.e., SH-chitosan hydrogel;
[0062] S43. Mixing the hydrogel precursor solution: Mix 0.5 mL of MAP peptide solution and 0.5 mL of SH-chitosan solution, gently vortex at room temperature, and let stand. Due to the Michael addition reaction between maleimide groups and thiol groups, the solution will gradually form a hydrogel precursor solution, namely SH-chitosan-MAP hydrogel, within about 30 min.
[0063] Note: Subsequent operations should be performed as soon as possible after mixing to avoid premature gelation;
[0064] Preparation of S44, SVF-hydrogel composite system: 1×10 7 One SVF cell (1 mL cell suspension) was gently mixed with the hydrogel precursor solution in S43 to obtain the final chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel, namely SVF@SH-chitosan-MAP hydrogel.
[0065] Note: The mixing process should be gentle to avoid damaging the cells.
[0066] SEM morphology and C / N / O / S elemental EDS (energy-dispersive X-ray diffraction) patterns of SH-chitosan hydrogel are shown below. Figure 4 As shown, the hydrogel morphology is relatively disordered and the fibrous network structure is not uniform. The EDS image shows that it contains sulfur, indicating that the hydrogel has been successfully thiolized.
[0067] The elemental distribution of SH-chitosan hydrogel is shown in Table 1:
[0068] Table 1
[0069] element Line type wt% Wt % Sigma At% C K-line system 46.28 0.30 53.04 N K-line system 7.70 0.43 7.57 O K-line system 45.58 0.29 39.21 S K-line system 0.44 0.03 0.19 Total 100.00 100.00
[0070] SEM morphology and EDS plots of C / N / O / S elements of SH-chitosan-MAP hydrogel are shown below. Figure 5 As shown in the figure, the morphology of SH-chitosan-MAP hydrogel shows that the fibrous network structure is more uniform, the pore size of the hydrogel fibrous network structure is about 25 μm, and there are a few protrusions on the inner surface of the fiber pores, which is conducive to cell adhesion and provides a better place for SVF adhesion. As can be seen from the EDS figure, the elemental content is almost unchanged compared with SH-chitosan hydrogel.
[0071] The elemental distribution of SH-chitosan-MAP hydrogel is shown in Table 2.
[0072] Table 2
[0073] element Line type wt% Wt % Sigma At% C K-line system 46.75 0.35 53.54 N K-line system 7.56 0.52 7.42 O K-line system 45.12 0.34 38.80 S K-line system 0.56 0.04 0.24 Total 100.00 100.00
[0074] FTIR (Fourier Transform Infrared Spectroscopy) of SH-chitosan hydrogel and SH-chitosan-MAP hydrogel, as shown below Figure 6 As shown, it can be seen that 2550 cm -1 A characteristic SH peak appears nearby at 1590 cm⁻¹. -1 The weakening or disappearance of the amino peak at the point indicates that SH-chitosan was successfully prepared.
[0075] XPS (X-ray photoelectron spectroscopy) of SH-chitosan hydrogel, such as Figure 7 As shown, the appearance of the characteristic peak of S2p (163-164 eV) indicates that the SH-chitosan hydrogel was successfully prepared.
[0076] XPS of SH-chitosan-MAP hydrogel Figure 8 As shown, MAP did not alter the original structure of the thiolated chitosan hydrogel.
[0077] The rheological diagrams of SH-chitosan hydrogel (a) and SH-chitosan-MAP hydrogel (b) are shown below. Figure 9As shown, SH-chitosan-peptide hydrogel is more stable and has better gel toughness than SH-chitosan hydrogel.
[0078] Mussel adhesive protein biomimetic peptides endow the hydrogel with strong adhesive ability, enabling it to adhere firmly to the cartilage surface and prevent SVF loss; as a carrier of SVF, the hydrogel can fix it in the joint cavity, prolong its residence time, and exert a continuous therapeutic effect.
[0079] Both chitosan and mussel adhesive protein biomimetic peptides have good biocompatibility and will not cause immune rejection. The RGD sequence in the peptides can promote cell adhesion, and chitosan provides a good cell growth microenvironment, which is beneficial to the colonization, survival and function of cells in SVF.
[0080] Chitosan itself has a certain cartilage repair effect. When combined with SVF, it can more effectively promote cartilage regeneration. The hydrogel precursor solution has good fluidity and can be injected into the joint cavity through a minimally invasive procedure, which is simple to perform.
[0081] Example 2
[0082] This embodiment provides a method for preparing a chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel:
[0083] The preparation method of S1, mussel adhesive protein biomimetic polypeptide is as follows:
[0084] The sequence “GGGRGDSPPP**YDK(DOPA)YDK(DOPA)**K-NH2” was designed by modifying the ε-amino group of lysine (K) with a maleimide group, where DOPA is 3,4-dihydroxyphenylalanine and RGD is arginine R-glycine G-aspartic acid D sequence.
[0085] The Fmoc solid-phase peptide synthesis method was adopted, using Wang resin and Fmoc-protected amino acids as monomers. The peptide chain was gradually extended through steps such as deprotection, condensation, and washing. HBTU / HOBt was selected as the condensing agent, and the condensation time was 2 hours.
[0086] After the peptide synthesis was completed, the peptide was cleaved using a TFA / TIS / H2O (volume ratio of 95:2.5:2.5) mixed solution and purified by reversed-phase high-performance liquid chromatography to obtain a peptide with a purity greater than 95%.
[0087] Finally, the purified peptide was dissolved in a buffer solution containing maleimide-NHS ester (excess) for modification of the maleimide group. After the reaction was completed, it was purified again and freeze-dried at -35 ℃ for 36 h to obtain the final mussel adhesive protein biomimetic peptide.
[0088] The sequence design of MAP peptides, which includes DOPA and RGD sequences, is key to achieving adhesion and cell adhesion.
[0089] S2. Preparation of thiolized chitosan:
[0090] In this embodiment, the chitosan is purchased medical-grade chitosan with a degree of deacetylation ≥90% and a molecular weight of 150kDa;
[0091] Chitosan was dissolved in a 0.5% (w / v) acetic acid solution to prepare a 1% (w / v) solution.
[0092] Add Triton B, with a molar ratio of Triton B to chitosan of 1:15, and stir the reaction at room temperature for 12 hours.
[0093] After the reaction was completed, the pH was adjusted to 2.0 using 1M HCl solution, and then dialyzed in deionized water for 36 h using a dialysis bag with a molecular weight cutoff of 3500 Da, with the deionized water being changed every 8 h.
[0094] Finally, the dialyzed solution was freeze-dried at -35 °C for 36 h to obtain thiolated chitosan;
[0095] S3. Preparation of SVF:
[0096] In this embodiment, the source of SVF was selected as rat autologous adipose tissue, such as subcutaneous fat from the abdomen or thigh.
[0097] Under aseptic conditions, fat aspiration was performed using standard tumescent anesthesia. The aspirated adipose tissue was washed three times with PBS to remove blood cells and free lipid droplets. Then, 0.075% (w / v) type I collagenase solution was added, and the mixture was incubated at 37 °C with shaking for 60 min for enzymatic digestion. After digestion, the mixture was centrifuged at 1200 g for 10 min to remove the upper lipid droplets and undigested tissue. The supernatant was discarded, and the cell pellet was resuspended in PBS. The cell suspension was filtered through a 100 μm cell sieve to remove tissue debris. The mixture was centrifuged at 400 g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in PBS, and the washing was repeated twice. The number of SVF cells was counted using a hemocytometer, and the cell concentration was adjusted to 1 × 10⁶ cells / mL using DMEM / F12 cell culture medium. 7 cells / mL, to obtain SVF;
[0098] S4. Preparation of composite hydrogel:
[0099] S41. Preparation of MAP peptide solution: Dissolve 10 mg of MAP peptide in 0.5 mL of PBS (pH 7.4) to prepare a MAP peptide solution with a concentration of 20 mg / mL.
[0100] S42, Preparation of SH-Chitosan Solution: Dissolve 10 mg of SH-chitosan in 0.15 mL of 0.5% (v / v) acetic acid solution to prepare a SH-chitosan solution with a concentration of 20 mg / mL, i.e., SH-chitosan hydrogel;
[0101] S43. Mixing the hydrogel precursor solution: Mix 0.1 mL of MAP peptide solution and 0.5 mL of SH-chitosan solution, gently vortex at room temperature, and let stand. Due to the Michael addition reaction between maleimide groups and thiol groups, the solution will gradually form a hydrogel precursor solution, namely SH-chitosan-MAP hydrogel, within about 10 minutes.
[0102] Preparation of S44, SVF-hydrogel composite system: 1×10 7 One SVF cell (1 mL cell suspension) was gently mixed with the hydrogel precursor solution in S43 to obtain the final chitosan-mussel adhesive protein biomimetic peptide-SVF composite hydrogel.
[0103] Example 3
[0104] This embodiment provides a method for preparing a chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel:
[0105] The preparation method of S1, mussel adhesive protein biomimetic polypeptide is as follows:
[0106] The sequence “GGGRGDSPPP**YDK(DOPA)YDK(DOPA)**K-NH2” was designed by modifying the ε-amino group of lysine (K) with a maleimide group, where DOPA is 3,4-dihydroxyphenylalanine and RGD is arginine R-glycine G-aspartic acid D sequence.
[0107] The Fmoc solid-phase peptide synthesis method was adopted, using Wang resin and Fmoc-protected amino acids as monomers. The peptide chain was gradually extended through steps such as deprotection, condensation, and washing. HBTU / HOBt was selected as the condensing agent, and the condensation time was 2 hours.
[0108] After the peptide synthesis was completed, the peptide was cleaved using a TFA / TIS / H2O (volume ratio of 95:2.5:2.5) mixed solution and purified by reversed-phase high-performance liquid chromatography to obtain a peptide with a purity greater than 95%.
[0109] Finally, the purified peptide was dissolved in a buffer solution containing maleimide-NHS ester (excess) for modification of the maleimide group. After the reaction was completed, it was purified again and freeze-dried at -35 ℃ for 36 h to obtain the final mussel adhesive protein biomimetic peptide.
[0110] The sequence design of MAP peptides, which includes DOPA and RGD sequences, is key to achieving adhesion and cell adhesion.
[0111] S2. Preparation of thiolized chitosan:
[0112] In this embodiment, the chitosan is purchased medical-grade chitosan with a degree of deacetylation ≥90% and a molecular weight of 150kDa;
[0113] Chitosan was dissolved in a 2% (w / v) acetic acid solution to prepare a 3% (w / v) solution;
[0114] Add Triton B, with a molar ratio of Triton B to chitosan of 1:15, and stir the reaction at room temperature for 12 hours.
[0115] After the reaction was completed, the pH was adjusted to 6.0 using 1M HCl solution, and then dialyzed in deionized water for 54 h using a dialysis bag with a molecular weight cutoff of 3500 Da, with the deionized water being changed every 8 h.
[0116] Finally, the dialyzed solution was freeze-dried at -35 °C for 36 h to obtain thiolated chitosan;
[0117] S3. Preparation of SVF:
[0118] In this embodiment, the source of SVF was selected as rat autologous adipose tissue, such as subcutaneous fat from the abdomen or thigh.
[0119] Under aseptic conditions, fat aspiration was performed using standard tumescent anesthesia. The aspirated adipose tissue was washed three times with PBS to remove blood cells and free lipid droplets. Then, 0.075% (w / v) type I collagenase solution was added, and the mixture was incubated at 37 °C with shaking for 60 min for enzymatic digestion. After digestion, the mixture was centrifuged at 1200 g for 10 min to remove the upper lipid droplets and undigested tissue. The supernatant was discarded, and the cell pellet was resuspended in PBS. The cell suspension was filtered through a 100 μm cell sieve to remove tissue debris. The mixture was centrifuged at 400 g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in PBS, and the washing was repeated twice. The number of SVF cells was counted using a hemocytometer, and the cell concentration was adjusted to 1 × 10⁶ cells / mL using DMEM / F12 cell culture medium. 7 cells / mL, to obtain SVF;
[0120] S4. Preparation of composite hydrogel:
[0121] S41. Preparation of MAP peptide solution: Dissolve 10 mg of MAP peptide in 0.5 mL of PBS (pH 7.4) to prepare a MAP peptide solution with a concentration of 20 mg / mL.
[0122] S42, Preparation of SH-Chitosan Solution: Dissolve 30 mg of SH-chitosan in 0.5 mL of 2% (v / v) acetic acid solution to prepare a 60 mg / mL SH-chitosan solution, i.e., SH-chitosan hydrogel;
[0123] S43. Mixing the hydrogel precursor solution: Mix 1 mL of MAP peptide solution and 0.5 mL of SH-chitosan solution, gently vortex at room temperature, and let stand. Due to the Michael addition reaction between maleimide groups and thiol groups, the solution will gradually form a hydrogel precursor solution, namely SH-chitosan-MAP hydrogel, within about 50 minutes.
[0124] Preparation of S44, SVF-hydrogel composite system: 1×10 7 One SVF cell (1 mL cell suspension) was gently mixed with the hydrogel precursor solution in S43 to obtain the final chitosan-mussel adhesive protein biomimetic peptide-SVF composite hydrogel.
[0125] Example 4
[0126] The following comparative experiments are conducted on the finished products prepared according to the above experimental embodiments to verify their technical effectiveness;
[0127] 1. Establishment of a rat model of osteoarthritis.
[0128] 1.1 Animal model establishment: An OA model was established using adult male SD rats by cutting the anterior cruciate ligament and medial meniscus.
[0129] 1.2 In this experiment, all animals were randomly divided into 4 groups.
[0130] 1) Normal group: Normal rats;
[0131] 2) OA group: PBS solution only;
[0132] 3) SVF group: SVF prepared in S3 of Example 1;
[0133] 4) SVF@SH-chitosan-MAP hydrogel group: SVF@SH-chitosan-MAP hydrogel prepared in S44 of Example 1;
[0134] 2. Collection, Micro-CT, embedding and slide preparation of knee joint specimens.
[0135] 2.1 The knee joints of rats were removed at week 8.
[0136] 2.2 The knee joints collected in 2.1 were subjected to micro-CT scans to reconstruct the three-dimensional structure of the joint. The results are as follows: Figure 10 As shown.
[0137] 2.3 Results: SVF and SVF@SH-chitosan-MAP hydrogel showed good therapeutic effects on osteoarthritis induced by anterior cruciate ligament and medial meniscus resection in rats. The femoral condyles of rats in the SVF@SH-chitosan-MAP hydrogel group were smoother, with no obvious osteophyte formation observed. This indicates that SVF@SH-chitosan-MAP hydrogel has a better therapeutic effect than SVF.
[0138] 2.4 Fix in 10% formalin for 24 h, rinse with running water for 2 min, add 10% EDTA decalcification solution, decalcify for 4-6 weeks until the bone tissue can be pierced by a needle without resistance, dehydrate with graded alcohol, clear with xylene, soak in paraffin for 3 hours, embed, cut into 4μm thick sections, and dewax and hydrate the sections.
[0139] 2.5 Safranin-Fixed Green Staining Procedure:
[0140] 1) Dewaxing and hydration of sections
[0141] 2) Perform staining according to the kit instructions.
[0142] 3) Dehydrate and clear the slices.
[0143] 4) Mount the slide with neutral resin and photograph it. The results are as follows: Figure 11 As shown.
[0144] 2.6 Results: SVF and SVF@SH-chitosan-MAP hydrogel showed good therapeutic effects on osteoarthritis induced by anterior cruciate ligament and medial meniscus resection in rats. Cartilage defects were observed on the articular surfaces of the SVF group, and synovial hyperplasia was observed in the joint space.
[0145] The joint surface of the SVF@SH-chitosan-MAP hydrogel group was smoother, and no obvious defects were observed, indicating that the SVF@SH-chitosan-MAP hydrogel had a better therapeutic effect than SVF.
[0146] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.
Claims
1. A method for preparing a chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel, characterized in that, Includes the following steps: S1. Preparation of mussel adhesive protein biomimetic peptides: The Fmoc solid-phase peptide synthesis method was adopted, using Wang resin and Fmoc-protected amino acids as monomers. The peptide chains of the open peptides were gradually extended through deprotection, condensation, and washing. The peptides were cut and purified to obtain purified peptides. The purified peptides were dissolved in buffer solution for modification. After the reaction was completed, the peptides were purified again and freeze-dried to obtain mussel adhesive protein biomimetic peptides. S2. Preparation of thiolated chitosan: Chitosan was dissolved in acetic acid solution to obtain a mixed solution. Triton B was added to the mixed solution and the mixture was stirred at room temperature. After the reaction was completed, the pH value of the solution was adjusted, and then dialyzed in deionized water using a dialysis bag. The dialyzed solution was freeze-dried to obtain thiolated chitosan. S3. Preparation of SVF: Fat aspiration was performed using standard tumescent anesthesia. The aspirated adipose tissue was washed with PBS to remove blood cells and free lipid droplets. Then, type I collagenase solution was added, and the mixture was shaken in a water bath for enzymatic digestion. After digestion, the supernatant was removed by centrifugation once to remove the upper lipid droplets and undigested tissue. The supernatant was discarded, and the cell pellet was resuspended in PBS. The cell suspension was filtered through a cell sieve to remove tissue debris. The cell pellet was centrifuged a second time to remove the supernatant, and the cell pellet was resuspended in PBS. After washing, the number of SVF cells was counted using a hemocytometer, and the cell concentration was adjusted with DMEM / F12 cell culture medium to obtain SVF. S4. Preparation of composite hydrogel: S41. Preparation of mussel adhesive protein biomimetic polypeptide solution: Dissolve mussel adhesive protein biomimetic polypeptide in PBS to prepare mussel adhesive protein biomimetic polypeptide solution. S42. Preparation of thiolated chitosan solution: Dissolve thiolated chitosan in acetic acid solution to prepare thiolated chitosan solution; S43. Mixing of hydrogel precursor solution: Mix mussel adhesive protein biomimetic polypeptide solution and thiolized chitosan solution, gently vortex at room temperature to mix, and let stand to form hydrogel precursor solution. Preparation of S44 and SVF-hydrogel composite system: SVF was gently mixed with the hydrogel precursor solution in S43 to obtain the final chitosan-mussel adhesive protein biomimetic peptide-SVF composite hydrogel.
2. The method for preparing the chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel according to claim 1, characterized in that, The condensing agent used in S1 is HBTU / HOBt, the condensation time is 2 h, the solution used to cleave the polypeptide is a TFA / TIS / H2O solution with a volume ratio of 95:2.5:2.5, the purification method is reversed-phase high-performance liquid chromatography, the purity of the purified polypeptide is >95%, the buffer is maleimide-NHS ester, the freeze-drying temperature is -35℃, and the freeze-drying time is 36 h.
3. The method for preparing the chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel according to claim 1, characterized in that, The chitosan in S2 has a degree of deacetylation ≥90% and a molecular weight of 150 kDa. The concentration of the acetic acid solution is 0.5%–2%, the concentration of the mixed solution is 1%–3%, the molar ratio of Triton B to chitosan is 1:5–15, the stirring reaction time is 12–36 h, the solvent for adjusting the pH of the solution is a 1 mol / L HCl solution, the target pH of the adjusted solution is 2.0–6.0, the molecular weight cutoff of the dialysis bag is 3500 Da, the dialysis time is 36–54 h with deionized water replaced every 8 h, the freeze-drying temperature is -35 ℃, and the freeze-drying time is 36 h.
4. The method for preparing the chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel according to claim 1, characterized in that, The mass concentration of the type I collagenase solution in S3 is 0.075%, the water bath temperature is 37 ℃, the enzyme digestion time is 60 min, the centrifugal force of the first centrifugation is 1200 g, the centrifugation time is 10 min, the pore size of the cell sieve is 100 μm, the centrifugal force of the second centrifugation is 400 g, the centrifugation time is 5 min, and the cell concentration of the SVF is 1×10⁻⁶. 7 cells / mL.
5. The method for preparing the chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel according to claim 1, characterized in that, The ratio of mussel adhesive protein biomimetic polypeptide to PBS in S41 is 10 mg: 0.5 mL, the pH value of the PBS is 7.4, and the concentration of the mussel adhesive protein biomimetic polypeptide solution is 20 mg / mL.
6. The chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel according to claim 5, its preparation method and application, characterized in that, In S42, the ratio of thiolated chitosan to acetic acid solution is 10–30 mg: 0.5 mL, the concentration of acetic acid solution is 0.5%–2%, and the concentration of thiolated chitosan solution is 20–60 mg / mL.
7. The method for preparing the chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel according to claim 6, characterized in that, The ratio of the mussel adhesive protein biomimetic polypeptide solution and the thiolated chitosan solution in S43 is 0.1-1 mL:0.5 mL, and the standing time is 10-50 min.
8. The method for preparing the chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel according to claim 7, characterized in that, The amount of SVF used in S44 is 1 mL.
9. An application of the chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel as described in any one of claims 1-7, characterized in that, The chitosan-mussel adhesive protein biomimetic polypeptide-SVF composite hydrogel is used to promote cartilage regeneration.