Umbilical cord mesenchymal stem cell and application thereof in treating osteoarthritis

Abstract

An umbilical cord mesenchymal stem cell and application thereof in treating osteoarthritis. The application discloses a dual-response hydrogel drug delivery system for osteoarthritis treatment and application thereof, and relates to a synergistic design of a bifunctional fusion protein BFP, an epigenetic regulator TSA and a temperature-sensitive-enzyme response hydrogel. The bifunctional protein BFP contains a cartilage induction domain CIP-2 and an anti-inflammatory domain AIP-3, which are connected in series through a rigid connection peptide EAKAK, and can specifically activate the TGF-beta pathway and respond to MMP9 to release the anti-inflammatory fragment. The temperature-sensitive-enzyme dual-response hydrogel is composed of thiolated hyaluronic acid and poloxamer 407, and can rapidly gelate within 3.5±0.5 minutes at 37 DEG C, the gel transition point is 29.2±0.3 DEG C, and the synergistic release of BFP / TSA can be realized in the presence of MMP9, and the 72-hour release rate reaches 86.7%. Experiments prove that the system can make the SOX9 / COL2A1 gene expression of UC-MSCs increase by 6.77 times and 5.9 times respectively, the GAG deposition amount reaches 0.81 OD630, the ICRS score in the rat OA model increases by 267%, and the serum MMP-13 level decreases by 78.3%. Therefore, the technology of the application provides a new direction for the precise treatment of osteoarthritis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a dual-response hydrogel drug delivery system for the treatment of osteoarthritis and its application. BACKGROUND

[0002] Osteoarthritis (OA) is a chronic disease with degenerative changes in joint cartilage as its core, and the prevalence rate among people over 65 years old worldwide is as high as 10%-15%, which seriously affects the quality of life of patients and brings heavy social burden. Current clinical treatment methods have significant limitations: non-drug treatments (exercise therapy, physical therapy) are difficult to fundamentally intervene in the course of the disease; non-steroidal anti-inflammatory drugs and corticosteroids can provide short-term relief from pain and inflammation, but long-term use can cause adverse reactions such as gastrointestinal damage and osteoporosis; sodium hyaluronate intra-articular injection can only temporarily improve joint lubrication and cannot reverse cartilage damage; joint replacement is suitable for end-stage patients, but it has high surgical risk, high cost, and limited service life of the prosthesis.

[0003] Umbilical cord mesenchymal stem cells (UC-MSCs) have become a research hotspot in the field of osteoarthritis treatment due to their strong proliferation capacity, low immunogenicity, and multi-directional differentiation potential. Their mechanisms of action include differentiation into chondrocytes, secretion of growth factors to regulate the joint microenvironment, and inhibition of inflammatory response. However, there are still three major bottlenecks in existing technology. First, the differentiation efficiency needs to be improved. For example, using a single chondrocyte induction peptide combined with chondroitin sulfate, the in vitro chondrogenic differentiation efficiency is only improved by 40%, and it cannot overcome the inhibition of differentiation by the inflammatory microenvironment. Second, the mechanism of action is relatively single. Patent CN118766964A co-loads UC-MSCs and exosomes in a hydrogel, which prolongs the cell retention time, but does not effectively enhance the cell's own directional differentiation ability. Third, the delivery system has defects. The cell retention rate is less than 20% after intra-articular injection, and the targeting of subchondral bone is poor; while subchondral injection can increase the number of trabecular bone by 2.1 times to improve bone microstructure, but it is difficult to popularize clinically due to its complex operation.

[0004] A 2025 study shows that the progression of osteoarthritis is closely related to the overactivation of matrix metalloproteinases (MMP2 / 9 / 13) and ADAMTS4 / 5. UC-MSCs can delay cartilage degradation by down-regulating these proteinases, but existing research lacks a synergistic design of cell function. Even if UC-MSCs stored by cryopreservation can reduce the WOMAC pain score by 2.66 times with a 12-month effect, there is still no significant progress in structural repair, and MRI detection shows no statistically significant improvement in cartilage thickness. Therefore, developing a multi-dimensional strategy that integrates "high-efficiency differentiation induction", "inflammatory microenvironment regulation", and "precise slow-release delivery" is the key to breaking through the technical bottlenecks of UC-MSCs in the treatment of osteoarthritis. SUMMARY

[0005] The present application aims to provide a kind of umbilical cord mesenchymal stem cells and its application in treating osteoarthritis. Based on this, the present application constructs "dual-function protein-epigenetic regulator-smart hydrogel" ternary synergistic system, and realizes the precise treatment of osteoarthritis by the following innovative design:

[0006] Therefore, the present application discloses a kind of dual-function fusion protein BFP in one aspect, its amino acid sequence is as shown in SEQ ID NO:1. Wherein BFP is through rigid connection peptide EAKAK series cartilage induction domain CIP-2 with anti-inflammatory domain AIP-3. Wherein, CIP-2 includes cartilage induction peptide GPPGPQG and its C-terminal insertion TGF-β1 receptor binding motif KFRR, can activate TGF-β signal pathway to promote osteoblast migration and chondrogenesis;AIP-3 is based on IL-1Ra active fragment VEPKYC, its N-terminal connection flexible peptide GGGGS and MMP9 high response cleavage site PLGLAG, can be released anti-inflammatory active fragment in inflammatory microenvironment by MMP9 enzyme cutting. The design makes BFP have double functions of cartilage differentiation induction and inflammation responsive anti-inflammatory.

[0007] The present application also discloses a kind of temperature-sensitive-enzyme dual-response hydrogel delivery system and its construction. The delivery system takes 20% poloxamer 407 and 0.5% thiolated hyaluronic acid (HA-SH) as matrix, the degree of substitution of MMP9 recognition sequence PLGLAG in HA-SH is 5.2%±0.8%, and the thiolation rate reaches 18.5%±1.2%. The system is liquid (viscosity 120 cP) at 4 DEG C, and quickly gels at 37 DEG C within 3.5±0.5 minutes, and the gel transition point is 29.2±0.3 DEG C, which meets the physiological temperature gelation requirement. Through the dual mechanisms of temperature triggered physical gelation and MMP9 mediated enzyme cutting, the cumulative release rate of BFP reaches 86.7% within 72 hours in the environment containing MMP9, and only 14.8% without MMP9, to realize the precise controlled release of drug at inflammatory site.

[0008] On the basis of the above, the present application also discloses a kind of ternary synergistic system. Dual-response hydrogel carries UC-MSCs (1×10 7cells / mL) and synergistically delivered BFP (150 pg / mL) with TSA (50 nM) to form a ternary system of "functional protein-epigenetic regulation-smart carrier". TSA promoted the SOX9 / COL2A1 promoter chromatin openness by 4.3-fold through inhibiting HDAC activity, and synergistically promoted UC-MSCs chondrogenic differentiation with BFP. qPCR showed that SOX9 / COL2A1 gene expression was increased by 6.77-fold and 5.90-fold, respectively, and Alcian Blue staining confirmed that the GAG deposition amount reached 0.81 OD630. In the rat OA model, this system improved the ICRS score to 11.0±0.6, reduced the serum MMP-13 level by 78.3%, and inhibited the joint MMP9 activity by 83.2%, achieving synergistic treatment of cartilage structure repair and inflammatory microenvironment regulation.

[0009] The present technology realizes the double breakthroughs of cartilage repair and inflammation inhibition in osteoarthritis treatment through the ternary synergistic design of "dual functional protein-epigenetic regulator-smart hydrogel": the combination of dual functional protein BFP and TSA can increase the SOX9 / COL2A1 gene expression of UC-MSCs by 4.3-6.7-fold, and the GAG deposition amount reaches 0.81 OD630. In the rat OA model, the ICRS score is improved to 11.0±0.6, close to the normal joint level; the warm-sensitive-enzyme dual-responsive hydrogel quickly gels at 37°C (3.5±0.5 min) and realizes the specific release of BFP in the presence of MMP9 (72h release rate 86.7%), reducing the serum MMP-13 level by 78.3% and inhibiting the joint MMP9 activity by 83.2%. This system has cell compatibility (viability >95%) and synergistic therapeutic effect, breaking through the efficiency bottleneck of single therapy and providing a precise treatment plan for osteoarthritis.

[0010] The present technology builds a precise treatment system for osteoarthritis through multi-dimensional innovation: for the first time, a dual functional fusion protein BFP is designed, which combines a cartilage induction peptide with an MMP9-responsive anti-inflammatory peptide to realize the dual functions of differentiation induction and inflammation inhibition; an epigenetic regulator TSA is innovatively integrated to enhance the expression of stem cell differentiation-related genes by more than 4.3-fold through chromatin opening regulation; a warm-sensitive-enzyme dual-responsive hydrogel is constructed, which utilizes the temperature-triggered gelation property of poloxamer and the MMP9 enzyme-cleavage drug release mechanism of HA-SH to realize rapid gelation at 37°C and precise drug release at inflammatory sites; through the ternary synergy of "functional protein-epigenetic regulation-smart carrier", the efficiency bottleneck of traditional single factor therapy is broken, and synergistic therapeutic effect of cartilage repair and inflammation inhibition is achieved in the rat OA model, providing a new paradigm for osteoarthritis treatment. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 BFP / TSA / UC-MSCs dual-responsive hydrogel construction mechanism schematic diagram. DETAILED DESCRIPTION

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0014] Example 1: Design, preparation and test of BFP fusion protein

[0015] 1.1 Rational design of bifunctional fusion protein (BFP)

[0016] 1.1.1 Design of cartilage induction domain (CIP-2)

[0017] Based on the cartilage induction peptide GPPGPQG, a TGF-β1 receptor binding motif KFRR is inserted at the C-terminal to form "GPPGPQG-KFRR"; on the one hand, it promotes osteoblast migration and chondrogenesis, and on the other hand, it enhances the affinity with TGF-βRII.

[0018] 1.1.2 Design of anti-inflammatory domain (AIP-3)

[0019] Based on the IL-1Ra active fragment VEPKYC, a flexible linker GGGGS and a MMP9 highly responsive cleavage site PLGLAG are added at the N-terminal to form "PLGLAG-GGGGS-VEPKYC".

[0020] 1.1.3 Preparation of bifunctional fusion protein BFP (referred to as BFP)

[0021] CIP-2 and AIP-3 are connected in series through a rigid linker EAKAK to avoid domain interference, and the full-length SEQ ID NO: 1 is GPPGPQG-KFRR-EAKAK-PLGLAG-GGGGS-VEPKYC.

[0022] Chemical coupling method is used to prepare BFP, and the final obtained BFP (SEQ ID NO: 1) is sent to Shengong Bioengineering (Shanghai) Co., Ltd. for synthesis, with a purity of ≥95%, and is stored at -80°C for standby.

[0023] 1.2 Experimental test

[0024] The umbilical cord of healthy volunteers after full-term delivery was selected, and after the informed consent of the puerpera, the umbilical cord was cut into about 1-2 cm small pieces under sterile conditions, and the residual blood was repeatedly washed away with PBS buffer containing double antibiotics (penicillin 100 U / mL, streptomycin 100 μg / mL). The umbilical cord tissue was cut longitudinally, the blood vessels were peeled off, and the umbilical cord was cut into 1-2 mm 3 size tissue pieces, inoculated in culture bottles, and added with α-MEM medium containing 10% fetal bovine serum (FBS), 1% glutamine and double antibiotics, and placed in a 37°C, 5% CO2 incubator for culture. The culture medium was replaced every 3 days, and when the primary cells crawled out of the tissue pieces and fused to 80-90%, the cells were passaged with 0.25% trypsin-EDTA digestion solution, and the 3rd-5th generation UC-MSCs in the logarithmic growth phase were used for subsequent experiments.

[0025] 1.2.1 Cartilage induction activity detection

[0026] BFP was added to the cartilage induction medium of UC-MSCs, and UC-MSCs containing only the basic cartilage induction medium were used as the control group, and the group added with GPPGPQG polypeptide was used as the positive control group. After 21 days of culture, the expression of cartilage-specific genes was detected by quantitative PCR.

[0027] The results showed that the expression of SOX9 gene in the BFP group was 3.58 times that of the control group, and the expression of COL2A1 gene was 2.90 times that of the control group, while the expression of SOX9 gene in the positive control group was 2.8 times that of the control group, and the expression of COL2A1 gene was 2.5 times that of the control group. At the same time, alizarin red staining was used to detect the synthesis of proteoglycan in the extracellular matrix, and the absorbance value (OD595) of the BFP group was 0.85±0.06, which was significantly higher than that of the positive control group (0.62±0.05) and the control group (0.23±0.03).

[0028] 1.2.2 Anti-inflammatory activity detection

[0029] In the lipopolysaccharide (LPS)-induced inflammation model, BFP was co-cultured with UC-MSCs, and the levels of inflammatory factors in the cell culture supernatant were detected.

[0030] Results showed that compared with the LPS stimulation group, the IL-1β content in the BFP group decreased from (250.3 ± 18.5) pg / mL to (85.6 ± 7.2) pg / mL, and the TNF-α content decreased from (320.5 ± 22.3) pg / mL to (112.4 ± 9.8) pg / mL. By Western blot detection of key proteins in the inflammatory signaling pathway, it was found that the phosphorylation level of NF-κB p65 in the BFP group was significantly reduced, to 35% of the LPS stimulation group. Further verification of MMP9 response, in the inflammation model with the addition of exogenous MMP9, the release amount of anti-inflammatory active fragments in the BFP group increased by 2.3 times compared with the group without the addition of MMP9, and the inhibition effect of inflammatory factors increased by 40%.

[0031] 1.3 Experimental summary

[0032] In this embodiment, a bifunctional fusion protein BFP containing cartilage induction domain CIP-2 and anti-inflammatory domain AIP-3 was successfully constructed. The experimental results show that BFP can significantly enhance the cartilage induction differentiation ability of UC-MSCs, and its effect is better than that of single cartilage induction peptide; at the same time, BFP shows good anti-inflammatory activity in the inflammatory microenvironment, and has the characteristics of MMP9 response release, which can realize targeted anti-inflammatory. Compared with the existing patent technology, this design breaks through the limitation of using only natural cytokines and lacking intelligent response mechanism, and provides a more innovative and efficient strategy for the treatment of osteoarthritis, and lays a solid foundation for the subsequent research on UC-MSCs modified by BFP for the treatment of osteoarthritis.

[0033] Example 2: Synergistic effect of small molecule epigenetic modulator TSA

[0034] 2.1 Purpose of the experiment

[0035] To explore the molecular mechanism and synergistic effect of histone deacetylase inhibitor TSA (trichostatin A) on enhancing BFP-induced cartilage differentiation of UC-MSCs through epigenetic regulation.

[0036] 2.2 Experimental design as shown in Table 1

[0037] Table 1 Experimental design

[0038]

[0039] TSA concentration optimization: Through pre-experiment test of 25nM, 50nM, 100nM concentration, the SOX9 gene expression is the highest when the concentration is 50nM, and the cell activity is >95%.

[0040] BFP concentration: The previous experiment confirmed that 150 μg / mL is the best non-toxic concentration for cartilage differentiation induction.

[0041] 2.3 Detection index and method

[0042] (1) Chromatin opening detection (ATAC-qPCR): After 72 hours of cell culture, the cells were washed twice with pre-cooled PBS and 1 x 10 5 cells were collected. Then 50 μL of ATAC reaction buffer containing 2.5 mM MgCl2 and 10 μL of transposase were added, and the mixture was incubated at 37°C with shaking at 500 rpm for 30 minutes. After the reaction, the DNA was purified using the MinElute PCR purification kit, and the elution volume was 10 μL. Subsequently, qPCR detection was performed, and the reaction system was 20 μL, containing 10 μL SYBR Green, 0.5 μL primer and 5 μL DNA template. The reaction program was set as 95°C pre-denaturation for 30 seconds, followed by 40 cycles of 95°C denaturation for 5 seconds and 60°C annealing for 30 seconds.

[0043] (2) qPCR of cartilage-specific genes (Day 7): RNA was extracted by the TRIzol method. After extracting total RNA, its purity was detected by Nanodrop, and the OD 260 / 280 was required to be between 1.9-2.0. During reverse transcription, 2 μg of RNA was mixed with 5x PrimeScript Mix, and the reaction was first carried out at 37°C for 15 minutes, and then the reverse transcription process was completed by treating at 85°C for 5 seconds. Subsequently, the cDNA obtained by reverse transcription was used as a template for qPCR amplification of cartilage-specific genes (SOX9, COL2A1, ACAN, etc.), and β-actin was used as an internal reference gene.

[0044] (3) Alcian Blue staining (Day 14): After 14 days of cell culture, the cells were first fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then rinsed with 1% acetic acid twice. Subsequently, the cells were stained with 1% Alcian Blue (pH 2.5) for 2 hours, and then rinsed with distilled water until the background was colorless. Finally, the cells were washed with 3% acetic acid to elute the staining agent, and the absorbance value of the eluate at OD 630 nm was measured by a microplate reader to quantitatively detect the enrichment of glycosaminoglycans (GAGs) in the extracellular matrix.

[0045] (4) CCK-8 cytotoxicity detection (Day 3): On the 3rd day after cell treatment, 10 μL of CCK-8 reagent was added to each well, and incubated at 37°C for 2 hours. Then the absorbance was measured at 450 nm by a microplate reader, and the cell activity was calculated by the formula (absorbance of experimental group / absorbance of control group) x 100%, to evaluate the effect of TSA, BFP, etc. on cell activity and determine their biocompatibility.

[0046] 2.4 Experimental results

[0047] (1) Chromatin openness: The chromatin openness of SOX9 and COL2A1 promoter region in each group was detected by ATAC-qPCR technology. The results showed (Table 2) that the promoter openness of SOX9 and COL2A1 in G4 group was increased by 4.32 times and 3.90 times compared with G1 group, respectively. The increase in G2 group was only 1.82 times and 1.62 times, and that in G3 group was 2.51 times and 2.11 times, respectively. The increase in G4 group was significantly higher than that in G2 group and G3 group (p < 0.05), indicating that TSA can effectively open the chromatin structure of SOX9 and COL2A1 genes by inhibiting the activity of HDAC (histone deacetylase) and synergize with BFP to create favorable conditions for gene transcription.

[0048] Table 2 ATAC-qPCR results (relative chromatin openness, n = 3)

[0049]

[0050] (2) Gene expression: The expression levels of cartilage-specific genes SOX9, COL2A1 and ACAN were detected by qPCR at day 7 of culture. The data showed (Table 3) that the expression of SOX9 gene in G4 group was increased by 6.77 times compared with G1 group, and the expression of COL2A1 gene was increased by 5.90 times, and the expression of ACAN gene was increased by 4.70 times. In contrast, the expression of each gene in G2 group using BFP alone and G3 group using TSA alone was lower than that in G4 group. This result fully confirmed that TSA can enhance the transcriptional activation of BFP on cartilage differentiation-related genes, and the synergistic effect of the two significantly promotes the expression of key genes in the process of UC-MSCs differentiation into chondrocytes.

[0051] Table 3 qPCR results (Day 7, n = 3)

[0052]

[0053] (3) Matrix synthesis: The deposition amount of glycosaminoglycan (GAG) in extracellular matrix was quantitatively detected by Alcian Blue staining. The results showed (Table 4) that the GAG accumulation in G4 group reached 0.81 (OD630 nm), which was about 5.8 times of that in G1 group (0.14). Compared with G2 group (0.43) and G3 group (0.39), the GAG deposition amount in G4 group also showed a significant advantage (p < 0.05). This data indicates that the synergistic effect of TSA and BFP not only promotes the expression of cartilage differentiation-related genes, but also effectively promotes the synthesis of extracellular matrix of chondrocytes, which is helpful for the repair and reconstruction of cartilage tissue.

[0054] Table 4 Alcian Blue staining for quantitative detection of GAG deposition (OD630 nm)630 nm, n = 3)

[0055] Group Replicate 1 Replicate 2 Replicate 3 Mean G1 0.13 0.15 0.14 0.14 G2 0.40 0.45 0.42 0.43 G3 0.37 0.39 0.41 0.39 G4 0.78 0.83 0.80 0.81

[0056] (4) Cytotoxicity: CCK-8 assay was used to detect cell viability at day 3 after cell treatment. The results showed (Table 5) that when the concentration of TSA was 50 nM, the cell viability of G4 group (TSA combined with BFP) remained at 98.2%, which had no statistically significant difference compared with G1 group (100%) (p > 0.05), and the cell viability of G4 group was slightly higher than that of G2 group (96.8%) and G3 group (95.5%). This result confirmed that the combination of TSA (50 nM) and BFP did not significantly reduce cell viability, and the combination of the two had good biocompatibility and played a synergistic therapeutic role under the premise of ensuring normal physiological functions of cells.

[0057] Table 5 CCK-8 cell viability (Day 3, n = 3)

[0058] Group Activity value 1 (%) Activity value 2 (%) Activity value 3 (%) Mean (%) G1 99.5 102.3 98.2 100.0 G2 94.3 98.5 97.6 96.8 G3 93.7 96.8 96.0 95.5 G4 96.7 99.3 98.6 98.2

[0059] 2.5 Experimental summary

[0060] TSA synergizes with BFP to enhance the chondrogenic differentiation ability of UC-MSCs through epigenetic regulation (chromatin opening), which is manifested as up-regulation of gene expression, increase of matrix synthesis, and no obvious cytotoxicity. This combined strategy provides a new method to break through the bottleneck of stem cell differentiation efficiency.

[0061] Example 3: Construction of thermo-enzyme dual-responsive hydrogel and BFP sustained-release study

[0062] 3.1 Material ratio and preparation process

[0063] (1) Synthesis of thiolated hyaluronic acid (HA-SH): In terms of raw material ratio, 5 g of hyaluronic acid with a molecular weight of 100 kDa was used, together with 6.2 mmol of 3-mercaptopropionic acid, 7.5 mmol of EDC and 7.5 mmol of NHS; during the reaction, the above raw materials were placed in an acetic acid buffer with a pH of 5.5, and stirred continuously at room temperature for 12 hours; after the reaction was completed, purification was performed by dialysis for 72 hours using a dialysis bag with a molecular weight cutoff of 10 kDa, followed by freeze-drying treatment; finally, the degree of substitution was determined using the Ellman reagent method, and the thiolation rate was measured to be 18.5% ± 1.2%.

[0064] (2) The preparation process of thermo-enzyme dual-responsive hydrogel is shown in Table 6 and Figure 1 .

[0065] Table 6 Preparation steps of thermo-enzyme dual-responsive hydrogel and UC-MSCs composition

[0066]

[0067] The key parameters of the system are as follows: temperature sensitivity is shown as liquid at 4°C, viscosity is 120 cP, and rapid gelation occurs at 37°C with a gelation time of 3.5 ± 0.5 minutes; in terms of enzyme cutting site, HA-SH contains the MMP9 recognition sequence PLGLAG, and the degree of substitution is 5.2% ± 0.8%.

[0068] 3.2 Gel performance detection

[0069] 3.2.1 Temperature-sensitive rheological characterization: temperature scanning (1°C / min, strain 1%, frequency 1 Hz) was performed using an MCR302 rheometer to systematically study the viscoelastic behavior transition of the hydrogel. The results show that:

[0070] (1) 25°C liquid phase: storage modulus G' = 85 ± 5 Pa, loss modulus G" = 120 ± 8 Pa, G" > G', the material shows fluid characteristics dominated by viscosity;

[0071] (2) 37°C gel phase: G' increases by 17.6 times to 1500 ± 100 Pa, G" decreases to 150 ± 12 Pa, G' is significantly higher than G" (G' / G" > 10), forming an elastic-dominated three-dimensional network structure;

[0072] (3) Gel transition temperature (Tgel): the temperature at which G' = G" is determined by the cross-point method to be 29.2 ± 0.3°C, which is close to the body temperature, ensuring rapid gelation in vivo.

[0073] 3.2.2 MMP9-responsive drug release: the temperature-enzyme dual-responsive hydrogel can rapidly gel in situ within 3.5 ± 0.5 minutes at 37°C physiological temperature. And in the inflammatory microenvironment containing MMP9, the cumulative release rate of BFP within 72 hours reaches 86.7% (only 14.8% in the MMP9-free group), through the dual mechanisms of temperature-triggered physical gelation and MMP9-mediated enzymatic drug release, precise drug control at the inflammation site is achieved, effectively avoiding the toxic side effects of systemic administration. As shown in Table 7.

[0074] Table 7 MMP9-responsive release (ELISA quantification, n = 3)

[0075]

[0076] 3.3 Cell compatibility and differentiation evaluation

[0077] 3.3.1 Cell viability and scaffold biocompatibility: Live / Dead staining (Day 1) showed that the percentage of viable cells (Calcein-AM+) in the G4 group (gel + UC-MSCs) was 96.3 ± 2.1%, which was not significantly different from the 2D culture group (97.1 ± 1.8%) (p > 0.05), indicating that the hydrogel system had no significant inhibitory effect on the activity of UC-MSCs and had good cell compatibility.

[0078] 3.3.2 Cartilage differentiation induction effect: Quantitative analysis of COL2A1 expression by immunofluorescence (Day 14) found that the COL2A1 positive cell rate in the G4 group (TSA + BFP co-release) was 78.5 ± 5.3%, which was significantly higher than that in the G2 group (BFP single release, 45.2 ± 4.1%) and the G3 group (TSA single release, 39.8 ± 3.7%) (p < 0.01), confirming that the synergistic release of TSA and BFP in the hydrogel system can effectively promote the expression of cartilage-specific proteins by UC-MSCs.

[0079] In summary, Live / Dead staining confirmed that the viability of UC-MSCs in the hydrogel was over 95%, which was not significantly different from 2D culture; immunofluorescence showed that the COL2A1 positive cell rate in the TSA + BFP co-release group was 78.5%, which was significantly higher than that in the BFP single release group (45.2%) and the TSA single release group (39.8%), indicating that the carrier system not only maintains high cell activity, but also enhances the expression efficiency of cartilage differentiation-related proteins through synergistic delivery.

[0080] 3.4 Gene expression (qPCR, Day 14)

[0081] qPCR data showed (Table 8) that the slow release of TSA and BFP in the hydrogel could increase the expression of SOX9 / COL2A1 genes by 1.8-2.3 times compared to the single drug group. This combined strategy of "intelligent delivery system + epigenetic modulator + functional polypeptide" breaks through the efficiency bottleneck of single therapy through the synergistic effect of chromatin opening regulation and differentiation induction, and verifies the additive advantage of multi-dimensional treatment scheme.

[0082] Table 8 qPCR data for gene expression (Day 14, n = 3)

[0083]

[0084] The above experimental results collectively show that the temperature-sensitive-enzyme dual-responsive hydrogel not only can maintain the high activity of UC-MSCs, but also can enhance the cartilage differentiation ability of cells through synergistic delivery of TSA and BFP, providing a new carrier system for osteoarthritis treatment that has both biocompatibility and functional effectiveness.

[0085] 3.5 Experimental summary

[0086] The successfully constructed temperature-sensitive-enzyme dual-responsive hydrogel has good biocompatibility and intelligent drug release characteristics, can synergistically deliver TSA and BFP, and significantly improves the chondrogenic differentiation efficiency of UC-MSCs through the dual mechanisms of epigenetic regulation and functional polypeptide induction. The system provides an innovative delivery platform for stem cell combination therapy of osteoarthritis.

[0087] Example 4: Study on the therapeutic effect of BFP / TSA / UC-MSCs dual-responsive hydrogel in a rat osteoarthritis model

[0088] 4.1 Animal model construction and grouping

[0089] 4.1.1 Experimental animals: SPF male SD rats (n = 36) were selected for the experiment, aged 6 weeks, weighing 220-250 g. The feeding environment was controlled at a temperature of 22 ± 2°C and a humidity of 50 ± 10%, with a 12-hour light-dark cycle, and the rats were allowed to freely eat and drink water.

[0090] 4.1.2 OA model preparation: The rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (40 mg / kg), and the right knee joint area was disinfected with iodophor and alcohol. During the operation, a 1.5 cm incision was made on the anteromedial side of the knee joint, the quadriceps tendon and patella were bluntly separated to expose the joint cavity, and the anterior cruciate ligament (ACL) femoral attachment point was cut off under a microscope (×10) to ensure complete disconnection of the ligament. The sham operation group only exposed the joint cavity without cutting the ACL. Postoperative management, intramuscular injection of penicillin (40,000 U / kg) for 3 consecutive days to prevent infection, rats were single-caged and allowed to freely move to avoid cross-infection.

[0091] 4.2 Grouping and treatment plan, a total of 4 groups were set up in this experiment, 9 rats in each group:

[0092] (1) Model group: ACLT operation was performed, and 0.1 mL PBS was injected into the knee joint cavity on the 7th day after operation as a blank control;

[0093] (2) Comparison group: ACLT operation was also performed, and on the 7th day after operation, a hydrogel based on the formula of patent CN118766964A was injected, which contained UC-MSCs 1 × 10 7 cells / mL and exosomes 200 μg / mL, the dose was calculated according to the body weight of the rats;

[0094] (3) Invention group: On the 7th day after ACLT operation, dual-responsive hydrogel was injected, which contained UC-MSCs 1 × 10 7Hydrogel drug loading was calculated according to the joint cavity volume (about 0.1 mL) based on the following concentrations: MSCs 1000 cells / mL, BFP 150 μg / mL, and TSA 50 nM.

[0095] (4) Sham group: only expose the joint cavity without cutting the ACL, inject 0.1 mL PBS at 7 days post-operation, as normal joint control.

[0096] The administration operation was performed at 7 days post-operation, using a 1 mL sterile syringe to puncture the knee joint cavity at a 45° needle angle, with a 5 mm needle depth to complete the injection. During the follow-up period, the rats were weighed every week, and the gait abnormality was observed using the Basso scoring system. The rats were sacrificed at 4, 8, and 12 weeks post-operation (3 rats per group per time point), for subsequent detection and analysis.

[0097] 4.3 Detection items and methods

[0098] 4.3.1 Histological scoring (based on ICRS standard)

[0099] In terms of sample preparation, the right knee joint was removed immediately after the rats were sacrificed, and fixed with 4% paraformaldehyde for 48 hours to stabilize the tissue morphology. Subsequently, 10% EDTA was used for decalcification treatment for 2 weeks to ensure that the hard tissue was softened for subsequent operations. After completing the decalcification, the sample was paraffin-embedded, and continuous sections were taken along the sagittal plane, with a section thickness of 5 μm. To ensure the representativeness of the detection, every 50 μm, one section was selected, and finally 6 sections were obtained for each sample for staining analysis.

[0100] In the staining and scoring process, H&E and Safranin O-fast green staining were used, and according to the ICRS scoring standard, the cartilage tissue was quantitatively evaluated from three dimensions with a score of 0-4. In terms of cartilage thickness, 4 represents full layer loss, and 0 represents complete full layer; in terms of surface integrity, 4 corresponds to deep cracks reaching the radiating layer, and 0 is smooth without cracks; the matrix staining intensity is based on the Safranin O staining, 4 is the disappearance of staining, and 0 is deep and uniform staining. The scoring process was completed by two experienced pathologists independently, and the Kappa coefficient of double-blind scoring reached 0.89, indicating good consistency of the scores.

[0101] 4.3.2 ELISA detection of inflammatory factors

[0102] A 500 μL blood sample was collected via the tail vein and incubated at 4°C for 1 hour to allow for coagulation and stratification. The sample was then centrifuged at 3000 rpm for 15 minutes to separate the serum, which was transferred to a new EP tube and stored at -80°C for later testing. Commercially available MMP-13 ELISA Kits and Rat IL-6 ELISA Kits were used. During the experiment, 100 μL each of the standard (with a 5-point gradient) and the sample were added to the wells of the ELISA plate and incubated at 37°C for 2 hours. TMB chromogenic buffer was then added, and the plate was incubated in the dark for 15 minutes. Finally, the absorbance was read at 450 nm using an ELISA reader. For data processing, background values ​​from blank wells were first subtracted, and then a four-parameter equation was used to fit the standard curve. The goodness of fit R0 was measured. 2 >0.99, ensuring the accuracy of the test results.

[0103] 4.3.3 In vivo MMP9 imaging

[0104] Using PerkinElmer's MMPsense TM The 680FAST probe was prepared at a concentration of 2 nmol / kg and dissolved in PBS buffer. The imaging device was an IVIS Spectrum system, with excitation / emission wavelengths set to 674 / 700 nm. The probe was injected into rats via the tail vein, and a 2-hour waiting period was allowed for sufficient enrichment of the probe in the MMP9 active region. Rats were then anesthetized with 2% isoflurane and fixed in a prone position on the imaging stage. Exposure parameters were set as follows: f / stop = 1, bin value = 8, and exposure time = 60 seconds. Imaging was then performed. For quantitative analysis, a region of interest (ROI) with a diameter of 5 mm was defined in the knee joint region, using photosensits / s / cm. 2 / sr is used as the unit of fluorescence intensity to quantify MMP9 activity.

[0105] 4.4 Experimental Results

[0106] 4.4.1 ICRS score (Table 9): The total ICRS score of the present invention group reached 11.0±0.6, which was 267% higher than that of the model group. The cartilage thickness, surface integrity and matrix staining were significantly improved, approaching the level of the sham surgery group, which confirms that the dual-response hydrogel system can effectively promote the repair of cartilage structure in the OA model, and the effect is better than that of the control group.

[0107] Table 9 ICRS Scoring Results (Week 8, n=6)

[0108]

[0109] Statistics: One-way ANOVA, *p<0.01 vs model group, **p<0.001 vs model group (Tukey’s post hoc)

[0110] 4.4.2 Serum MMP-13 levels (Table 10): The serum MMP-13 levels of the inventive group decreased to 8.2 ± 0.9 ng / mL at week 8, which was 78.3% lower than that of the model group, and continued to maintain a low level, indicating that the dual-responsive hydrogel effectively inhibited the release of matrix degradation enzymes caused by joint inflammation by releasing TSA and BFP in a sustained manner, and the anti-inflammatory effect was significantly better than that of the comparative groups.

[0111] Table 10 Serum MMP-13 levels (ng / mL, n = 3)

[0112] Time point Model group Control group Invention group Sham group Week 4 31.2±2.5 18.5±1.8* 12.1±1.3* 10.3±1.1 Week 8 39.7±3.2 15.8±1.5* 8.2±0.9* 9.5±0.8 Week 12 45.2±3.8 14.7±1.2* 9.8±1.1* 10.2±1.0

[0113] Statistics: Repeated measures ANOVA, *p<0.01, **p<0.001 vs model group

[0114] 4.4.3 In vivo MMP9 fluorescence intensity (Table 11): The MMP9 activity fluorescence intensity of the knee joint of the inventive group was 83.2% lower than that of the model group, close to the level of the sham operation group, confirming that the dual-responsive hydrogel releases drugs in response to MMP9 enzyme cutting in an inflammatory microenvironment, specifically inhibits the activity of intra-articular matrix metalloproteinases, and blocks the inflammation-degradation vicious cycle.

[0115] Table 11 In vivo MMP9 fluorescence intensity (week 8, n = 3)

[0116]

[0117] 4.5 Histological images and mechanism analysis

[0118] Typical Safranin O-Fast Green staining performance: The cartilage layer thickness of the inventive group recovered to 85% of normal, the GAG retention rate was >90%, and the cells were arranged regularly, indicating that the UC-MSCs differentiated into chondrocytes in the dual-responsive hydrogel and synthesized a large amount of extracellular matrix; the GAG loss of the model group was >70%, and the repair of the comparative group was incomplete, further verifying the synergistic repair advantage of the inventive system.

[0119] Histological observation confirmed that the dual-responsive hydrogel provided a three-dimensional scaffold through temperature-sensitive gelation, combined with the synergistic drug release of TSA and BFP, promoted the differentiation of UC-MSCs into chondrocytes, significantly improved the synthesis and retention of extracellular matrix, and achieved functional repair of cartilage tissue.

[0120] 4.6 Experimental summary

[0121] In this study, we combined temperature-sensitive enzyme dual-responsive hydrogel, epigenetic regulator TSA and functional polypeptide BFP for the first time, and used UC-MSCs to treat rat OA model. The results showed that this system realized the time and space controllable release of TSA and BFP through the dual mechanisms of MMP9 enzyme cleavage response and temperature-sensitive in situ gelation, synergistically promoted the chondrogenic differentiation of UC-MSCs, improved the ICRS score by 267%, reduced the serum MMP-13 level by 78.3%, inhibited the joint MMP9 activity by 83.2%, and the retention rate of cartilage matrix GAG was more than 90%.

[0122] The innovation points are: 1) Constructing a triple synergistic treatment strategy of "intelligent delivery system + epigenetic regulation + functional induction", breaking through the efficiency bottleneck of traditional single factor treatment; 2) Using dual-responsive hydrogel to realize precise drug release at the inflammation site, reducing systemic toxic side effects; 3) Integrating the three-dimensional culture microenvironment of UC-MSCs to improve cell adhesion and differentiation efficiency. This system provides an innovative treatment paradigm for the clinical transformation of osteoarthritis, which has biocompatibility, targeting and synergistic effect.

[0123] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, which are all included in the protection scope of the present application.

Claims

1. A bifunctional fusion protein BFP, characterized in that, The amino acid sequence of the BFP is shown as SEQ ID NO:

1.

2. A UC-MSCs composition, characterized in that, The composition comprises the following ingredients: (1) the bifunctional fusion protein BFP of claim 1, at a concentration of 150 μg / mL; (2) the epigenetic modulator trichostatin A (TSA), at a concentration of 50 nM; (3) a suspension of UC-MSCs in an amount of 1 x 10 7 cells / mL; (4) a thermo-enzyme dual-responsive hydrogel.

3. The composition of claim 2, wherein, The thermo-enzyme dual-responsive hydrogel is a thermo-enzyme responsive matrix consisting of 20% poloxamer 407 and 0.5% thiolated hyaluronic acid.

4. The composition of claim 3, wherein, The thiolated hyaluronic acid contains the MMP9 recognition sequence PLGLAG, with a degree of substitution of 5.2%±0.8%.

5. The composition of claim 2, wherein, The thermo-enzyme dual-responsive hydrogel is in a liquid state at 4℃, with a viscosity of 120 cP, a gelation time of 3.5±0.5 minutes at 37℃, and a gel transition point of 29.2±0.3℃.

6. Use of the composition of claim 2 in the preparation of a drug for the treatment of osteoarthritis.

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