Umbilical cord mesenchymal stem cell and application thereof in treatment of osteoarthritis

Abstract

The invention relates to umbilical cord mesenchymal stem cells and application thereof in treatment of osteoarthritis. The invention discloses a double-response hydrogel drug delivery system for osteoarthritis treatment and application of the double-response hydrogel drug delivery system, and relates to collaborative design of bifunctional fusion protein BFP, an epigenetic regulator TSA and temperature-sensitive-enzyme response hydrogel. The bifunctional protein BFP comprises a cartilage induction domain CIP-2 and an anti-inflammatory domain AIP-3 which are connected in series through rigid connecting peptide EAKAK, and can specifically activate a TGF-beta pathway and respond to MMP9 to release an anti-inflammatory fragment. The temperature-sensitive-enzyme double-response hydrogel is composed of thiolated hyaluronic acid and poloxamer 407, rapid gelation is conducted within 3.5 + / -0.5 min at 37 DEG C, the gel transformation point is 29.2 + / -0.3 DEG C, synergistic release of BFP / TSA is achieved in the presence of MMP9, and the release rate reaches 86.7% within 72 h. Experiments prove that by means of the system, SOX9 / COL2A1 gene expression of UC-MSCs can be improved by 6.77 times and 5.9 times respectively, the GAG deposition amount reaches 0.81 OD630, the ICRS score in a rat OA model is improved by 267%, and the serum MMP-13 level is reduced by 78.3%. Therefore, the technology provided by the invention provides a new direction for precise treatment of osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a dual-responsive hydrogel drug delivery system for treating osteoarthritis and applications thereof. Background Art

[0002] Osteoarthritis (OA) is a chronic disease characterized by degenerative changes in joint cartilage. Globally, the prevalence of OA in people over 65 is as high as 10%-15%, severely impacting patients' quality of life and placing a heavy social burden. Current clinical treatments have significant limitations: non-drug therapies (exercise therapy, physical therapy) struggle to fundamentally intervene in the disease's course; while non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids can provide short-term pain and inflammation relief, long-term use can lead to adverse reactions such as gastrointestinal damage and osteoporosis; intra-articular injections of sodium hyaluronate only temporarily improve joint lubrication and cannot reverse cartilage damage; and joint replacement surgery, reserved for terminally ill patients, carries significant risks, high costs, and a limited lifespan.

[0003] Umbilical cord mesenchymal stem cells (UC-MSCs) have become a research hotspot in the treatment of osteoarthritis due to their strong proliferation capacity, low immunogenicity, and multidirectional differentiation potential. Their mechanisms of action include differentiation into chondrocytes, secretion of growth factors to modulate the joint microenvironment, and suppression of inflammatory responses. However, existing technologies still face three key bottlenecks. First, differentiation efficiency urgently needs to be improved. For example, using a single chondroitin-inducing peptide combined with chondroitin sulfate only increases chondrogenic differentiation efficiency by 40% in vitro and fails to overcome the inhibitory effects of the inflammatory microenvironment on differentiation. Second, the mechanism of action is relatively simple. Patent CN118766964A co-delivers UC-MSCs and exosomes into a hydrogel. While this method prolongs cell retention time, it fails to effectively enhance the cells' own directed differentiation capacity. Third, the delivery system is flawed. The cell retention rate after intra-articular injection is less than 20%, resulting in poor targeting of the subchondral bone. While subchondral injection can increase trabecular bone count by 2.1-fold and improve bone microarchitecture, its complex procedure hinders its widespread clinical application.

[0004] The latest research in 2025 shows that the progression of osteoarthritis is closely related to the overactivation of matrix metalloproteinases (MMP2 / 9 / 13) and ADAMTS4 / 5. Although UC-MSCs can delay cartilage degradation by downregulating these proteases, existing studies lack a synergistic enhancement design for cell function. Even though a single injection of cryopreserved UC-MSCs can reduce the WOMAC pain score by 2.66 times, showing a 12-month efficacy, there is still no significant progress in structural repair, and MRI examinations show no statistical improvement in cartilage thickness. Therefore, the development of a multidimensional strategy that integrates "efficient differentiation induction", "inflammatory microenvironment regulation" and "precise sustained-release delivery" is the key to breaking through the technical bottleneck of UC-MSCs in the treatment of osteoarthritis. Summary of the Invention

[0005] The present invention aims to provide umbilical cord mesenchymal stem cells and their use in the treatment of osteoarthritis. Based on this, the present invention constructs a ternary synergistic system of "bifunctional protein-epigenetic modulator-intelligent hydrogel" to achieve precise treatment of osteoarthritis through the following innovative designs:

[0006] Therefore, on one hand, the present invention discloses a bifunctional fusion protein BFP, whose amino acid sequence is shown in SEQ ID NO: 1. BFP is a chondrogenic domain CIP-2 and an anti-inflammatory domain AIP-3 connected in series via a rigid linker peptide EAKAK. CIP-2 contains the chondrogenic peptide GPPGPQG and the TGF-β1 receptor binding motif KFRR inserted at its C-terminus, which can activate the TGF-β signaling pathway to promote osteoblast migration and cartilage formation; AIP-3 is based on the IL-1Ra active fragment VEPKYC, whose N-terminus is connected to the flexible peptide GGGGS and the MMP9 highly responsive cleavage site PLGLAG, which can be cleaved by MMP9 in the inflammatory microenvironment to release the anti-inflammatory active fragment. This design enables BFP to have both the dual functions of cartilage differentiation induction and inflammation-responsive anti-inflammatory.

[0007] The present invention also discloses a thermosensitive-enzyme dual-responsive hydrogel delivery system and its construction. The delivery system is based on 20% poloxamer 407 and 0.5% thiolated hyaluronic acid (HA-SH). The degree of substitution of the 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 at 4°C (viscosity 120cP) and rapidly gels within 3.5 ± 0.5 minutes at 37°C. The gel transition point is 29.2 ± 0.3°C, which meets the requirements of physiological temperature gelation. Through the dual mechanism of temperature-triggered physical gelation and MMP9-mediated enzymatic drug release, the cumulative release rate of BFP reaches 86.7% within 72 hours in an MMP9-containing environment, and only 14.8% in the absence of MMP9, achieving precise controlled release of drugs in inflammatory sites.

[0008] On the basis of the above, the present invention also discloses a ternary synergistic system. Dual-responsive hydrogel loaded with UC-MSCs (1×10 7cells / mL) and co-delivered BFP (150μg / mL) and TSA (50nM), forming a "functional protein-epigenetic regulation-intelligent carrier" ternary system. TSA increased the chromatin accessibility of the SOX9 / COL2A1 promoter by 4.3-fold by inhibiting HDAC activity, and synergistically promoted the chondrogenic differentiation of UC-MSCs with BFP. qPCR showed that SOX9 / COL2A1 gene expression increased by 6.77-fold and 5.90-fold, respectively, and Alcian Blue staining confirmed GAG deposition at 0.81 OD630. In a rat OA model, this system increased the ICRS score to 11.0±0.6, reduced serum MMP-13 levels by 78.3%, and inhibited joint MMP9 activity by 83.2%, achieving synergistic treatment of cartilage structural repair and inflammatory microenvironment regulation.

[0009] This technology, through a ternary synergistic design of "bifunctional protein-epigenetic modulator-smart hydrogel," achieves a dual breakthrough in cartilage repair and inflammation suppression in the treatment of osteoarthritis. The bifunctional protein BFP combined with TSA increased SOX9 / COL2A1 gene expression in UC-MSCs by 4.3-6.7 times, GAG deposition reached 0.81 OD630, and ICRS scores in rat OA models increased to 11.0±0.6, approaching the level of normal joints. The thermosensitive-enzyme dual-responsive hydrogel rapidly gelled at 37°C (3.5±0.5 minutes) and specifically released BFP in the presence of MMP9 (86.7% release rate over 72 hours), reducing serum MMP-13 levels by 78.3% and inhibiting MMP9 activity in joints by 83.2%. This system combines cytocompatibility (viability >95%) with synergistic therapeutic effects, surpassing the efficiency bottleneck of monotherapy and providing a precise treatment option for osteoarthritis.

[0010] This technology constructs a precise treatment system for osteoarthritis through multi-dimensional innovation: for the first time, a bifunctional fusion protein BFP is designed, which connects cartilage-inducing peptides and MMP9-responsive anti-inflammatory peptides in series to achieve the dual functions of differentiation induction and inflammation inhibition; the epigenetic regulator TSA is innovatively integrated to enhance the expression of stem cell differentiation-related genes by more than 4.3 times through chromatin open regulation; a thermosensitive-enzyme dual-responsive hydrogel is constructed, which utilizes the temperature-triggered gelation properties of poloxamer and the MMP9 enzymatic cleavage and drug release mechanism of HA-SH to achieve rapid gelation at 37°C and precise drug release at the inflammatory site; through the three-way synergy of "functional protein-epigenetic regulation-intelligent carrier", it breaks through the efficiency bottleneck of traditional single-factor treatment, and achieves the synergistic effect of cartilage repair and inflammation inhibition in the rat OA model, providing a new paradigm for the treatment of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0013] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

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

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

[0016] 1.1.1 Design of the chondrogenic inductive domain (CIP-2)

[0017] Based on the chondroitin-inducing peptide GPPGPQG, the TGF-β1 receptor binding motif KFRR is inserted into its C-terminus to form "GPPGPQG-KFRR", which promotes osteoblast migration and chondrogenesis while enhancing affinity with TGF-βRII.

[0018] 1.1.2 Anti-inflammatory domain (AIP-3) design

[0019] Based on the IL-1Ra active fragment VEPKYC, the flexible connecting peptide GGGGS and the MMP9 highly responsive cleavage site PLGLAG were added to its N-terminus to form "PLGLAG-GGGGS-VEPKYC".

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

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

[0022] BFP was prepared by chemical coupling method. The final BFP (SEQ ID NO: 1) was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis with a purity of ≥95% and stored at -80°C for future use.

[0023] 1.2 Experimental test

[0024] The umbilical cords of healthy volunteers who had given birth at full term were selected. After informed consent from the mothers, the umbilical cords were cut into small pieces of about 1-2 cm under sterile conditions and repeatedly rinsed with PBS buffer containing double antibiotics (penicillin 100 U / mL, streptomycin 100 μg / mL) to remove residual blood. The umbilical cord tissue was cut longitudinally, the blood vessels were stripped, and the pieces were cut into 1-2 mm pieces. 3 Tissue fragments of different sizes were seeded into culture flasks and cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and double-antibody antibodies at 37°C in a 5% CO2 incubator. The medium was changed every 3 days. When primary cells emerged from the tissue fragments and reached 80-90% confluence, they were passaged using 0.25% trypsin-EDTA digestion solution. UC-MSCs at passages 3-5 in the logarithmic growth phase were used for subsequent experiments.

[0025] 1.2.1 Cartilage induction activity assay

[0026] BFP was added to the chondrogenic induction medium of UC-MSCs. A control group of UC-MSCs cultured with basal chondrogenic induction medium alone served as the control, while a group supplemented with GPPGPQG peptide served as the positive control. After 21 days of culture, the expression of cartilage-specific genes was assessed by quantitative PCR.

[0027] The results showed that the expression of the SOX9 gene in the BFP group was 3.58 times that of the control group, and the expression of the COL2A1 gene was 2.90 times that of the control group. In the positive control group, the expression of the SOX9 gene was 2.8 times that of the control group, and the expression of the COL2A1 gene was 2.5 times that of the control group. At the same time, Alcian blue staining was used to detect the synthesis of proteoglycans in the extracellular matrix. 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] The results showed that compared with the LPS-stimulated 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. Western blot analysis of key proteins in the inflammatory signaling pathway revealed a significant decrease in the phosphorylation level of NF-κB p65 in the BFP group, decreasing to 35% of that in the LPS-stimulated group. Further validation of MMP9 responsiveness revealed that in an inflammatory model with exogenous MMP9 addition, the release of anti-inflammatory active fragments in the BFP group increased by 2.3 times compared to the group without MMP9 addition, and the inhibitory effect of inflammatory factors increased by 40%.

[0031] 1.3 Experimental Summary

[0032] In this example, a bifunctional fusion protein, BFP, containing the chondrogenic domain CIP-2 and the anti-inflammatory domain AIP-3, was successfully constructed through design. Experimental results showed that BFP can significantly enhance the chondrogenic differentiation ability of UC-MSCs, with an effect superior to that of a single chondroinductive peptide. Furthermore, BFP exhibits good anti-inflammatory activity in the inflammatory microenvironment and possesses MMP9-responsive release properties, enabling targeted anti-inflammatory responses. Compared with existing patented technologies, this design breaks through the limitations of using only natural cytokines and lacking intelligent response mechanisms, providing a more innovative and efficient strategy for the treatment of osteoarthritis and laying a solid foundation for subsequent research on the treatment of osteoarthritis with BFP-modified UC-MSCs.

[0033] Example 2: Synergistic Effect of Small Molecule Epigenetic Regulator TSA

[0034] 2.1 Experimental Purpose

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

[0036] 2.2 Experimental design is shown in Table 1

[0037] Table 1 Experimental design

[0038]

[0039] TSA concentration optimization: Through preliminary experiments, 25nM, 50nM, and 100nM concentrations were tested. At 50nM, the SOX9 gene expression level was the highest and the cell activity was >95%.

[0040] BFP concentration: Previous experiments have confirmed that 150 μg / mL is the optimal non-toxic concentration for inducing chondrogenic differentiation.

[0041] 2.3 Detection indicators and methods

[0042] (1) Chromatin open detection (ATAC-qPCR): After culturing cells in each group for 72 hours, they were washed twice with pre-cooled PBS and 1×10 5 cells. 50 μL of ATAC reaction buffer containing 2.5 mM MgCl2 and 10 μL of transposase were then 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. qPCR detection was then performed with a 20 μL reaction system containing 10 μL SYBR Green, 0.5 μL primers, and 5 μL DNA template. The reaction program was set to 95°C pre-denaturation for 30 seconds, followed by 40 cycles (95°C denaturation for 5 seconds, 60°C annealing for 30 seconds).

[0043] (2) Cartilage-specific gene qPCR (Day 7): RNA was extracted using the TRIzol method, and the purity of the total RNA was tested using Nanodrop. The OD 260 / 280 The pH value was between 1.9 and 2.0. For reverse transcription, 2 μg of RNA was mixed with 5× PrimeScript Mix, incubated at 37°C for 15 minutes, and then incubated at 85°C for 5 seconds to complete the reverse transcription process. Subsequently, the reverse-transcribed cDNA was used as a template for qPCR amplification of cartilage-specific genes (such as SOX9, COL2A1, and ACAN), using β-actin as an internal reference gene.

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

[0045] (4) CCK-8 cytotoxicity assay (Day 3): On the third day after cell treatment, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance was then measured at 450 nm using a microplate reader. Cell viability was calculated as (absorbance of the experimental group / absorbance of the control group) × 100%. This was used to evaluate the effects of TSA, BFP, and other treatments on cell viability and determine their biocompatibility.

[0046] 2.4 Experimental Results

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

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

[0049]

[0050] (2) Gene expression: qPCR was performed to detect the expression levels of cartilage-specific genes SOX9, COL2A1, and ACAN on the 7th day of culture. The data showed (Table 3) that the expression of SOX9 gene in the G4 group increased by 6.77 times relative to that in the G1 group, the expression of COL2A1 gene increased by 5.90 times, and the expression of ACAN gene increased by 4.70 times. In contrast, the expression of each gene in the G2 group using BFP alone and the G3 group using TSA alone was lower than that in the G4 group. This result fully confirmed that TSA can enhance the transcriptional activation effect of BFP on cartilage differentiation-related genes, and the synergistic effect of the two significantly promoted 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: Alcian Blue staining was used to quantitatively detect the deposition of glycosaminoglycans (GAGs) in the extracellular matrix. The results showed (Table 4) that the cumulative amount of GAGs in the G4 group reached 0.81 (OD630 nm), which was approximately 5.8 times that of the G1 group (0.14). Compared with the single-drug treatment groups of G2 group (0.43) and G3 group (0.39), the GAG ​​deposition amount in the G4 group also showed a significant advantage (p < 0.05). This data shows 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 cartilage extracellular matrix, which is conducive to the repair and reconstruction of cartilage tissue.

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

[0055] Group Repeat 1 Repeat 2 Repeat 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: The CCK-8 method was used to detect cell activity on the third day after cell treatment. The results showed (Table 5) that when the TSA concentration was 50 nM, the cell activity of the G4 group (TSA combined with BFP) remained at 98.2%, which was not statistically significant compared with the G1 group (100%) (p>0.05), and the cell activity of the G4 group was slightly higher than that of the G2 group (96.8%) and the G3 group (95.5%). This result confirms that the combined use of TSA (50 nM) and BFP does not significantly reduce cell activity. The combination of the two has good biocompatibility and exerts a synergistic therapeutic effect while ensuring the normal physiological function of the cells.

[0057] Table 5 CCK-8 cell activity (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 enhances the chondrogenic differentiation of UC-MSCs through epigenetic regulation (chromatin opening) in collaboration with BFP, resulting in upregulated gene expression and increased matrix synthesis without significant cytotoxicity. This combined strategy provides a new approach to overcome the bottleneck in stem cell differentiation efficiency.

[0061] Example 3: Construction of thermosensitive-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 100 kDa hyaluronic acid was used, along with 6.2 mmol 3-mercaptopropionic acid, 7.5 mmol EDC, and 7.5 mmol NHS. During the reaction, the raw materials were placed in an acetate buffer solution at pH 5.5 and stirred at room temperature for 12 hours. After the reaction, the product was purified by dialysis with a 10 kDa molecular weight cutoff for 72 hours, followed by lyophilization. The degree of substitution was finally determined using the Ellman reagent method, and the thiolation rate was found to be 18.5% ± 1.2%.

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

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

[0066]

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

[0068] 3.2 Gel performance test

[0069] 3.2.1 Thermosensitive rheological characterization: A temperature sweep (1°C / min, 1% strain, 1 Hz frequency) was performed using an MCR302 rheometer to systematically study the viscoelastic behavior transition of the hydrogel. The results showed:

[0070] (1) 25℃ liquid stage: storage modulus G'=85±5Pa, loss modulus G"=120±8Pa, G">G', the material exhibits viscosity-dominated fluid properties;

[0071] (2) Gel stage at 37°C: G' increased 17.6 times to 1500±100 Pa, G" decreased to 150±12 Pa, and G' was significantly higher than G" (G' / G">10), forming a three-dimensional network structure dominated by elasticity;

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

[0073] 3.2.2 MMP9-responsive drug release: This thermosensitive-enzyme dual-responsive hydrogel rapidly gelled in situ within 3.5 ± 0.5 minutes at a physiological temperature of 37°C. Furthermore, in an MMP9-containing inflammatory microenvironment, the cumulative BFP release rate reached 86.7% within 72 hours (compared to only 14.8% in the MMP9-free group). Through the dual mechanisms of temperature-triggered physical gelation and MMP9-mediated enzymatic cleavage, precise controlled drug release was achieved at the site of inflammation, effectively avoiding the toxic side effects of systemic drug administration. (See Table 7.)

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

[0075]

[0076] 3.3 Cytocompatibility and differentiation assessment

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

[0078] 3.3.2 Chondrogenic differentiation induction effect: When quantitatively analyzing COL2A1 expression by immunofluorescence (Day 14), it was found that the COL2A1 positive cell rate in the G4 group (TSA+BFP co-release) reached 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 in UC-MSCs.

[0079] In summary, Live / Dead staining confirmed that the viability of UC-MSCs in the hydrogel exceeded 95%, with no significant difference from 2D culture; immunofluorescence showed that the COL2A1-positive cell rate in the TSA+BFP co-release group reached 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 coordinated delivery.

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

[0081] qPCR data (Table 8) showed that sustained-release TSA and BFP via hydrogels increased SOX9 / COL2A1 gene expression by 1.8-2.3 times compared to the single-drug group. This combined strategy of "intelligent delivery system + epigenetic regulator + functional peptide" overcomes the efficiency bottleneck of single-therapy treatments through the synergistic effects of chromatin openness regulation and differentiation induction, validating the combined advantages of a multi-dimensional treatment approach.

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

[0083]

[0084] The above experimental results collectively indicate that the thermosensitive-enzyme dual-responsive hydrogel can not only maintain the high activity of UC-MSCs, but also enhance the chondrogenic differentiation ability of cells by synergistically delivering TSA and BFP, providing a new carrier system with both biocompatibility and functional effectiveness for the treatment of osteoarthritis.

[0085] 3.5 Experimental Summary

[0086] The thermosensitive-enzyme dual-responsive hydrogel successfully constructed in this example exhibits excellent biocompatibility and intelligent drug release properties. It can synergistically deliver TSA and BFP, significantly enhancing the chondrogenic differentiation efficiency of UC-MSCs through a dual mechanism of epigenetic regulation and functional peptide induction. This system provides an innovative delivery platform for combined stem cell therapy for 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 Sprague-Dawley rats (n=36), 6 weeks old, weighing 220-250 g, were used in this experiment. The rats were housed in a controlled environment with a temperature of 22±2°C, a humidity of 50±10%, and a 12-h light-dark cycle. They had free access to food and water.

[0090] 4.1.2 OA model preparation: Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (40 mg / kg), and the right knee joint area was disinfected with iodine tincture and alcohol. During the operation, a 1.5 cm incision was made on the anteromedial side of the knee joint, and the quadriceps tendon and patella were bluntly separated to expose the joint cavity. The anterior cruciate ligament (ACL) femoral attachment point was cut with microscissors under a microscope (×10) to ensure complete ligament detachment. In the sham operation group, only the joint cavity was exposed, and the ACL was not cut. In terms of postoperative management, intramuscular penicillin (40,000 U / kg) was injected for 3 consecutive days to prevent infection. Rats were housed in single cages and allowed to move freely to avoid cross infection.

[0091] 4.2 Grouping and treatment plan: This experiment set up 4 groups, with 9 rats in each group:

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

[0093] (2) Control group: The same ACLT surgery was performed and a hydrogel based on the patent CN118766964A formula containing 1×10 UC-MSCs was injected on the 7th day after surgery. 7 cells / mL and exosomes 200 μg / mL, the dosage was calculated based on the rat body weight;

[0094] (3) Invention group: On the 7th day after ACLT surgery, dual-responsive hydrogel containing 1×10 UC-MSCs was injected. 7cells / mL, BFP 150 μg / mL, and TSA 50 nM. The drug loading capacity of the hydrogel was calculated based on the volume of the joint cavity (approximately 0.1 mL);

[0095] (4) Sham operation group: Only the joint cavity was exposed without cutting the ACL. 0.1 mL PBS was injected on the 7th day after surgery to serve as a normal joint control.

[0096] Dosing was performed on the seventh day after surgery using a 1 mL sterile syringe inserted into the knee joint cavity at a 45° needle angle to a depth of 5 mm. Rats were weighed weekly during follow-up, and gait abnormalities were assessed using the Basso scoring system. Rats were sacrificed at four, eight, and 12 weeks after surgery (three rats per group at each time point) for subsequent testing and analysis.

[0097] 4.3 Testing items and methods

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

[0099] For sample preparation, the right knee joints were removed immediately after rat sacrifice and fixed in 4% paraformaldehyde for 48 hours to stabilize tissue morphology. Decalcification was then performed with 10% EDTA for two weeks to soften the hard tissue for subsequent manipulation. After decalcification, the specimens were paraffin-embedded and serially sectioned along the sagittal plane at a thickness of 5 μm. To ensure representativeness, sections were taken every 50 μm, resulting in six sections per specimen for staining and analysis.

[0100] During the staining and scoring process, H&E and Safranin O-Fast Green staining were used, and cartilage tissue was quantitatively assessed based on three dimensions, with a score of 0 to 4, according to the ICRS scoring system. For cartilage thickness, a score of 4 represents full-thickness loss, while a score of 0 indicates full-thickness integrity. For surface integrity, a score of 4 corresponds to deep cracks extending into the stratum radiatum, while a score of 0 indicates smooth, crack-free tissue. Matrix staining intensity was assessed based on Safranin O staining, with a score of 4 indicating absent staining and a score of 0 indicating deep, uniform staining. Scoring was performed independently by two experienced pathologists, and the double-blind scoring process achieved a Kappa coefficient of 0.89, indicating good consistency in scoring.

[0101] 4.3.2 ELISA detection of inflammatory factors

[0102] 500 μL of blood sample was collected through the tail vein and placed in a 4°C environment for 1 hour to allow the blood to fully coagulate and stratify; then centrifuged at 3000 rpm for 15 minutes, the serum was separated and transferred to a new EP tube, and stored in a -80°C ultra-low temperature refrigerator for testing. Commercial MMP-13ELISA Kit and Rat IL-6ELISA Kit were used. During the experiment, 100 μL of the standard (set 5-point gradient) and sample were added to the ELISA plate wells and incubated at a constant temperature of 37°C for 2 hours; then TMB colorimetric solution was added and color was developed in the dark for 15 minutes; finally, the absorbance value was read using an enzyme-linked reader at a wavelength of 450 nm. When processing the data, the background value of the blank well was first deducted, and then the standard curve was fitted using a four-parameter equation, and the goodness of fit R was 0.05. 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 the IVIS Spectrum system, and the excitation / emission wavelengths were set to 674 / 700 nm, respectively. The probe was injected into the rat through the tail vein. After 2 hours of injection, the probe was allowed to fully accumulate in the MMP9 active area. The rat was then anesthetized with 2% isoflurane and fixed in a prone position on the imaging table. The exposure parameters were set to 1 f / stop, 8 bins, and 60 seconds of exposure time to complete the imaging. For quantitative analysis, a region of interest (ROI) with a diameter of 5 mm was set in the knee joint area, and the image was acquired with a resolution of photons / s / cm 2 The fluorescence intensity was expressed as / sr, and the MMP9 activity was quantified.

[0105] 4.4 Experimental Results

[0106] 4.4.1ICRS score (Table 9): The total ICRS score of the present invention group reached 11.0±0.6, an increase of 267% compared with the model group. The cartilage thickness, surface integrity and matrix staining were significantly improved, close to the level of the sham operation group, confirming that the dual-responsive 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 score 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 level (Table 10): The serum MMP-13 level in the present invention group dropped to 8.2±0.9 ng / mL at week 8, a decrease of 78.3% compared with the model group, and continued to maintain a low level, indicating that the dual-responsive hydrogel effectively inhibited the release of matrix-degrading enzymes caused by joint inflammation by sustained-release TSA and BFP, and the anti-inflammatory effect was significantly better than that of the control group.

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

[0112] Time point Model Group comparison group The present invention group Sham operation 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 fluorescence intensity of MMP9 activity in the knee joints of the present invention group was reduced by 83.2% compared with the model group, approaching the level of the sham operation group, confirming that the dual-responsive hydrogel releases drugs in response to MMP9 enzymatic cleavage in the inflammatory microenvironment, specifically inhibiting the activity of matrix metalloproteinases in the joints and blocking the vicious cycle of inflammation-degradation.

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

[0116]

[0117] 4.5 Histological images and mechanism analysis

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

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

[0120] 4.6 Experimental Summary

[0121] This study, for the first time, combined a thermosensitive-enzyme dual-responsive hydrogel, the epigenetic regulator TSA, and the functional peptide BFP, using UC-MSCs to treat OA in a rat model. Results showed that this system, through a dual mechanism of MMP9-responsive drug release and thermosensitive in situ gelation, achieved spatiotemporally controlled release of TSA and BFP, synergistically promoting chondrogenic differentiation of UC-MSCs. This resulted in a 267% increase in ICRS scores, a 78.3% decrease in serum MMP-13 levels, an 83.2% inhibition of MMP9 activity in joints, and over 90% retention of cartilage matrix GAGs.

[0122] The innovations lie in: 1) constructing a triple synergistic therapeutic strategy of "intelligent delivery system + epigenetic regulation + functional induction," breaking through the efficiency bottleneck of traditional single-factor treatments; 2) utilizing a dual-responsive hydrogel to achieve precise drug release at the site of inflammation, minimizing systemic toxicity and side effects; and 3) integrating the three-dimensional culture microenvironment of UC-MSCs to enhance cell adhesion and differentiation efficiency. This system provides an innovative therapeutic paradigm for the clinical translation of osteoarthritis, combining biocompatibility, targeted therapy, and synergistic effects.

[0123] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

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

1.

2. A UC-MSCs composition, characterized in that: The composition comprises the following ingredients: (1) The bifunctional fusion protein BFP according to claim 1, at a concentration of 150 μg / mL; (2) epigenetic regulator TSA, at a concentration of 50 nM; (3) UC-MSCs suspension, with a content of 1×10 7 cells / mL; (4) Thermosensitive-enzyme dual-responsive hydrogel.

3. The composition according to claim 2, characterized in that The temperature-sensitive-enzyme dual-responsive hydrogel is a temperature-sensitive-enzyme responsive matrix composed of 20% poloxamer 407 and 0.5% thiolated hyaluronic acid.

4. The composition according to claim 3, characterized in that The thiolated hyaluronic acid contains the MMP9 recognition sequence PLGLAG, and the degree of substitution is 5.2%±0.8%.

5. The composition according to claim 2, characterized in that The temperature-sensitive-enzyme dual-responsive hydrogel is liquid at 4°C, has a viscosity of 120 cP, a gelation time of 3.5±0.5 minutes at 37°C, and a gel transition point of 29.2±0.3°C.

6. Use of the composition according to claim 2 in preparing a drug for treating osteoarthritis.

Images