A pH-responsive tendon-bone interface regeneration coaxial scaffold, preparation method and application

Abstract

The application provides a pH-responsive tendon-bone interface regeneration coaxial scaffold, a preparation method and application, the scaffold comprises an outer shell layer composed of silk fibroin with uniaxial orientation arrangement, an inner core layer composed of hyaluronic acid hydrogel loaded with von Willebrand factor, and the outer shell layer and the inner core layer form a coaxial scaffold; a surface modification layer comprising liposomes loaded with Peptide 5 covalently anchored on the surface of the outer shell layer through a Schiff base bond. The application also provides a preparation method of the scaffold and application of the scaffold in preparation of a medical device for treating tendon-bone interface defects.

Description

Technical Field

[0001] This invention relates to a pH-responsive tendon-bone interface regeneration coaxial scaffold, its preparation method, and its application, belonging to the field of biomedical materials technology. Background Technology

[0002] Tendon-bone interface injuries are common in orthopedics and are extremely difficult to manage clinically. Taking rotator cuff tears as an example, conservative treatment or arthroscopic suturing and bridging are still the mainstream approaches. The core of these methods lies in restoring the mechanical connection between the tendon and bone through physical means, but they do not address the biological degenerative process of the interface tissue itself, resulting in a persistently high rate of re-tears after surgery.

[0003] The natural regeneration process at the tendon-bone interface is extremely complex, and a key reason for repair failure lies in the severe mismatch between the pathological microenvironment formed at the injury site and the physiological conditions required for tissue regeneration. After injury, the accumulation of local metabolic byproducts leads to severe microenvironmental acidosis. This persistent acidic inflammatory environment causes proteins within resident cells to misfold, triggering a strong cellular stress response and initiating apoptosis, resulting in the depletion of the local endogenous progenitor cell pool before tissue regeneration has truly begun. Furthermore, the tendon-bone interface region itself suffers from poor blood supply, which also restricts nutrient supply and angiogenesis in the early stages of regeneration.

[0004] Current research in tissue engineering and biomaterials largely focuses on providing passive structural support or biomimetic physical morphology to the injury site. Some studies have also attempted to directly drive tissue regeneration through the delivery of single anabolic factors. However, these existing technologies cannot solve the problem of cell survival in the harsh local environment of acidity and high stress. Without alleviating the acidosis and high stress at the injury site, continuously and blindly pushing cells to differentiate and synthesize matrix will ultimately only result in disordered scar tissue, making it difficult to reconstruct the fibrocartilage interface with gradient mechanical function.

[0005] In summary, existing technologies lack an effective intervention method that can intelligently sense the pathological microenvironment, prioritize reversing cellular survival crises, and synergistically provide structural and vascular support to address the temporal needs of cell apoptosis driven by local microenvironment deterioration and interfacial multi-tissue regeneration. Summary of the Invention

[0006] The purpose of this invention is to provide a pH-responsive coaxial scaffold for tendon-bone interface regeneration, its preparation method, and its application, in order to solve the technical problem that existing scaffolds are unable to release active factors on demand and sequentially in an acidic inflammatory microenvironment and coordinate cell protection, immune regulation, and bipolar differentiation, thereby achieving functional regeneration of the tendon-bone interface.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a pH-responsive tendon-bone interface regeneration coaxial scaffold, the scaffold comprising:

[0009] The outer shell is composed of silk fibroin with a uniaxial orientation; the inner core is composed of hyaluronic acid hydrogel loaded with von Willebrand factor, and the outer shell and the inner core form a coaxial scaffold.

[0010] A surface modification layer comprising liposomes loaded with Peptide 5, covalently anchored to the surface of the outer shell layer via Schiff base bonds.

[0011] In conjunction with the first aspect, the sequence of Peptide 5 is VDCFLSRPTEKT.

[0012] Furthermore, the Schiff base bond breaks under acidic conditions with a pH not greater than 5.5, causing the liposomes loaded with Peptide 5 to dissociate and release Peptide 5.

[0013] Furthermore, the outer shell layer is a nanofiber structure, wherein the fiber diameter is 340 nm and the ultimate tensile strength is 12 MPa.

[0014] In a second aspect, the present invention provides a method for preparing the pH-responsive tendon-bone interface regenerative coaxial scaffold as described in any one of the first aspects, comprising the following steps:

[0015] Silk fibroin was dissolved in hexafluoroisopropanol to prepare a silk fibroin solution;

[0016] Hyaluronic acid and von Willebrand factor are dissolved in water to prepare a mixed solution containing hyaluronic acid and von Willebrand factor;

[0017] The silk fibroin solution and the mixed solution were electrospun through a coaxial needle, and the coaxial fibers with orientation were collected on a rotating drum receiver. The coaxial core-shell fiber scaffold was obtained by vacuum drying.

[0018] Liposomes loaded with Peptide 5 and having amino groups on their surface were prepared. The coaxial core-shell fiber scaffold was immersed in a reaction buffer containing the liposomes, so that the aldehyde groups on the surface of the silk fibroin reacted with the amino groups on the surface of the liposomes to form Schiff base bonds, thereby anchoring the Peptide 5-loaded liposomes to the fiber surface and obtaining the pH-responsive tendon-bone interface regeneration coaxial scaffold.

[0019] In conjunction with the second aspect, the electrospinning process parameters are further as follows: applied voltage 18 kV, receiving distance 15 cm, and roller speed 200 rpm.

[0020] Furthermore, the preparation of liposomes loaded with Peptide 5 and having amino groups on their surface includes: preparing liposomes loaded with Peptide 5 using a thin-film hydration method, and modifying them with octadecylamine by amino grouping.

[0021] Thirdly, the present invention provides a biomaterial composition comprising the pH-responsive tendon-bone interface regeneration coaxial scaffold as described in any one of the first aspects.

[0022] Fourthly, the present invention provides the use of the pH-responsive tendon-bone interface regeneration coaxial scaffold as described in any one of the first aspects in the preparation of a medical device for treating tendon-bone interface defects.

[0023] In conjunction with the fourth aspect, the tendon-bone interface defect further includes a rotator cuff tear.

[0024] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0025] This invention provides a pH-responsive tendon-bone interface regeneration coaxial scaffold with logic gating and spatiotemporal cascade release capabilities. The scaffold is constructed by forming a coaxial scaffold consisting of an outer shell composed of uniaxially oriented silk fibroin fibers and a core layer of hyaluronic acid hydrogel loaded with von Willebrand factor. Peptide 5-loaded liposomes are covalently anchored to the outer shell surface using Schiff base bonds, achieving stable scaffold operation under normal physiological conditions. When the scaffold is exposed to acidic conditions at a pH not exceeding 5.5, the Schiff base bonds break, triggering the dissociation of the Peptide 5-loaded liposomes and the release of Peptide 5, achieving a specific responsive release in acidic regions.

[0026] By releasing Peptide 5 to activate the DUSP5 molecular switch, the MAPK-mediated endoplasmic reticulum stress and apoptosis pathways are blocked, thus protecting the survival of endogenous progenitor cells in an acidic microenvironment.

[0027] The uniaxially oriented nanofibers in the outer shell provide topological guidance signals, enabling the directional alignment of rescued cells and the secretion of tendon-associated matrix. The hyaluronic acid hydrogel in the inner core layer sustains the release of von Willebrand factor, providing early vascularization support and synergistically promoting the regeneration of the fibrocartilage interface.

[0028] The synergistic effect of the coaxial structure and the surface covalent network enables the scaffold to achieve an ultimate tensile strength of 12 MPa, meeting the mechanical requirements of clinical surgical suturing.

[0029] This invention provides a method for preparing a pH-responsive tendon-bone interface regenerative coaxial scaffold, which employs a coaxial electrospinning combined with Schiff base click chemical grafting process. The steps are simple and controllable, achieving efficient anchoring of liposomes and promoting functional regeneration of the tendon-bone interface. Attached Figure Description

[0030] Figure 1 The fabrication and multidimensional structural characterization diagram of the biomimetic logic gated scaffold provided by this invention;

[0031] Figure 2 The diagram shows the kinetic programming and chemical fidelity verification of the pH-responsive "rescue" valve provided by this invention.

[0032] Figure 3 This diagram serves as a verification of the DUSP5-mediated molecular rescue mechanism and the blocking of the apoptosis cascade provided by the present invention.

[0033] Figure 4 An assessment diagram of immune homeostasis restoration, endoplasmic reticulum stress relief, and angiogenesis rescue during the microenvironment reprogramming process provided by this invention;

[0034] Figure 5 This diagram serves as a verification of the dual-system guidance and anabolism rebound effect during tissue reconstruction under stress, as provided by the present invention.

[0035] Figure 6 This invention provides a multi-omics panoramic verification diagram from transcriptome reconstruction to protein cascade blockade.

[0036] Figure 7 Imaging, molecular remodeling, and biomechanical recovery assessment diagrams for in vivo functional arrest repair provided by this invention;

[0037] Figure 8 A quantitative analysis diagram of the orientation degree of electrospun nanofibers provided by the present invention;

[0038] Figure 9 Live / dead staining assessment diagram of the in vitro basic cell compatibility of the scaffold provided by the present invention;

[0039] Figure 10 A quantitative statistical chart of the survival rate of cells cultured on each scaffold within 7 days, provided by this invention;

[0040] Figure 11 H&E staining images of various organs for in vivo systemic biosafety evaluation provided by this invention;

[0041] Figure 12 This is a quantitative analysis diagram of cell mortality rates in different groups under inflammatory stress, provided by the present invention.

[0042] Figure 13This invention provides a long-term evaluation diagram of lineage-specific differentiation of cells in an inflammatory microenvironment.

[0043] Figure 14 This invention provides a multidimensional quantitative verification diagram of early dual-lineage transcription factors.

[0044] Figure 15 An in vitro qualitative immunofluorescence evaluation diagram of angiogenesis activation provided by the present invention;

[0045] Figure 16 An in situ immunohistochemical analysis diagram of the regenerative microenvironment 8 weeks post-surgery, provided by this invention;

[0046] Figure 17 This is an evaluation diagram of early dual-lineage differentiation and matrix deposition at the rat rotator cuff regeneration interface 8 weeks post-surgery, provided by the present invention.

[0047] Figure 18 Immunohistochemical image of Bax expression at the regeneration arrest site 8 weeks post-surgery, provided by this invention;

[0048] Figure 19 Immunohistochemical analysis of Caspase-3 expression, the apoptosis executive factor, at 8 weeks post-surgery, as provided by this invention;

[0049] Figure 20 H&E staining assessment image of early macroscopic morphological reconstruction of tendon-bone interface at 8 weeks postoperatively, provided by the present invention.

[0050] Figure 21 H&E staining assessment diagram of tendon-bone interface morphology reconstruction at 12 weeks postoperatively, provided by the present invention.

[0051] Figure 22 Masson staining assessment of early collagen matrix deposition at the interface 8 weeks post-surgery, provided by this invention;

[0052] Figure 23 Masson staining assessment of late collagen matrix integration at 12 weeks post-surgery, provided by this invention;

[0053] Figure 24 SO-FG staining image of early chondrogenic and fibrocartilaginous matrix deposition at the interface 8 weeks post-surgery, provided by the present invention.

[0054] Figure 25 SO-FG staining assessment diagram of interface maturation fibrocartilage reconstruction 12 weeks postoperatively, provided by this invention. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and should not be used to limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional experimental methods; unless otherwise specified, the reagents and materials used are commercially available.

[0056] Example 1:

[0057] This embodiment describes the fabrication of a pH-responsive coaxial scaffold (SHLP), and the steps are as follows:

[0058] (1) Preparation of electrospinning precursor solution

[0059] Shell solution: The lyophilized silk fibroin (SF) powder extracted and purified from silkworm cocoons according to the established degumming scheme is dissolved in hexafluoroisopropanol (HFIP) solvent to prepare a shell solution with a mass-volume ratio of 10% (w / v). The solution is stirred at room temperature until completely dissolved and allowed to stand to remove bubbles for later use.

[0060] Core layer solution: Hyaluronic acid (HA) with a molecular weight of approximately 1.5 MDa was dissolved in deionized water, and recombinant rat von Willebrand factor (vWF) was added. The mixture was thoroughly mixed to prepare an aqueous solution containing vWF and HA at a mass-to-volume ratio of 5% (w / v).

[0061] (2) Coaxial electrospinning process

[0062] A biomimetic scaffold was fabricated using an ordered coaxial electrospinning apparatus. The prepared shell solution and HA / vWF core solution were respectively loaded into two independent injection pumps equipped with coaxial needles.

[0063] Spinning parameters were set as follows: the applied voltage was set to 18 kV, and the receiving distance between the receiver and the needle tip was set to 15 cm. To induce the nanofibers to exhibit a highly uniaxial orientation that mimics natural tendons, the roller speed was precisely set to 200 rpm. After collecting the spun film, it was placed in a vacuum drying oven to remove residual solvent, yielding a coaxial core-shell scaffold (SH). The hyaluronic acid in its core layer can absorb water to form a hydrogel under physiological conditions in vivo, which can be used to sustain the release of von Willebrand factor.

[0064] (3) Surface functionalization modification

[0065] Preparation of drug-loaded liposomes: Lecithin and cholesterol were mixed at a mass ratio of 4:1 using a thin-film hydration method, dissolved in an organic solvent, and then rotary evaporated to form a membrane. The lipid membrane was hydrated with a buffer containing peptide 5 (sequence: VDCFLSRPTEKT, purity >90%), and then sonicated to form uniformly sized liposomes loaded with peptide 5.

[0066] Amination modification: The liposomes prepared above were surface modified with octadecylamine (ODA) to give them primary amino groups on their surface.

[0067] (4) Schiff base click chemistry grafting

[0068] The SH scaffold was immersed in a reaction buffer solution, and the ODA-modified liposome suspension was added. The reaction was carried out at a suitable temperature, allowing the aldehyde groups exposed on the SF fiber surface (or aldehyde groups generated through activation treatment) to covalently react with the amino groups on the liposome surface, forming acid-sensitive Schiff base bonds. After the reaction, the scaffold was thoroughly washed with buffer solution to remove unbound free molecules, and then freeze-dried under vacuum to obtain the SHLP scaffold.

[0069] As controls, pure silk fibroin scaffolds (SF), coaxial core-shell scaffolds (SH), and coaxial scaffolds grafted with unloaded liposomes (SHL) were prepared simultaneously.

[0070] Characterization of scaffold morphology and physical properties;

[0071] The multidimensional structure of the above-mentioned hierarchical nanofiber scaffold was characterized, and the results are as follows: Figure 1 As shown. Figure 1 Image A shows a schematic diagram of the fabrication process of ordered coaxial electrospun nanofiber scaffolds, and image B is a scanning electron microscope image, which shows that the fibers in groups SF, SH, SHL and SHLP are all uniaxially oriented.

[0072] Figure 1 Transmission electron microscopy images of the C-type scaffold confirmed that the SH scaffold has a clear core-shell structure and that liposomes are grafted onto the fiber surface.

[0073] Figure 1 Image D is an atomic force microscopy topological image of the fiber surface before and after liposome functionalization, showing the formation of a hierarchical micro-nano rough structure after modification.

[0074] Figure 1 E in the figure is a statistical graph showing the change in porosity after the nanofiber scaffold is functionalized stepwise. The porosity decreases after the modification.

[0075] Figure 1 The graph in F is a statistical diagram showing the gradient change in fiber diameter distribution, with the fiber diameter increasing from approximately 180 nm in the SF group to approximately 340 nm in the SHLP group.

[0076] Figure 1 G represents the tensile strength and typical stress-strain mechanics test curves of each group of nanofiber scaffolds. The SHLP scaffold exhibits improved mechanical strength and toughness, with an ultimate tensile strength reaching 12 MPa, meeting the requirements for surgical applications. Figure 8This is a quantitative analysis diagram of the orientation degree of electrospun nanofibers. The diagram shows, from bottom to top, the quantitative fitting curves of the parallel orientation degree and polarization angle distribution of SF fibers, SH fibers, grafted liposome fibers and fully functional drug-loaded nanofibers along the stress axis on the surface of the roller-shaped collector. It confirms that the fiber orientation degree exceeds 80%, and this highly oriented structure provides anisotropic topological signals for tendon cells.

[0077] pH-responsive release kinetics and chemical structure confirmation;

[0078] Figure 2 The chemical composition and acid-sensitive release characteristics of the stent were verified. Figure 2 In the diagram, A is the molecular chemical structure of peptide Peptide 5, and B is the high-performance liquid chromatography (HPLC) chromatogram, showing that the purity of peptide Peptide 5 is higher than 90.88%. Figure 2 The mass spectrum of C confirmed the molecular structure of peptide Peptide 5.

[0079] Figure 2 In the middle section, D is a bar chart of quantitative analysis of scaffold surface roughness, surface area difference, and maximum roughness. E and F are the pH-dependent cumulative release kinetic curves of liposomes and peptide 5 under different pH conditions, respectively, showing a pH-dependent biphasic release characteristic: the release amount is extremely low at physiological pH 7.4, and the Schiff base bond undergoes protonation and breakage under acidic conditions at pH 5.5, triggering a burst release of peptide 5 within 10 hours, which is highly matched with the acute inflammatory time window.

[0080] Figure 2 G represents the relative weight degradation rate curve of each group of functionalized stents over time in vitro. The stents exhibit a moderate degradation rate within 25 days, ensuring a long-term supply of core factor vWF during the regeneration phase.

[0081] Figure 2 The middle H to K sections are high-resolution X-ray photoelectron spectroscopy (XPS) C1s spectra, in which... Figure 2 The surface of the SHLP scaffold from K showed characteristic C=N Schiff base bond signals, confirming the successful formation of covalent bonds. Figure 2 L and Figure 2 M represents the characteristic Raman spectra of each group of scaffolds. Figure 2 In the figure, N represents the XPS full-spectrum elemental map of the surface of each group of scaffolds. Figure 2 The values ​​in O represent the Fourier transform infrared spectra of each scaffold group, collectively confirming the successful recombination of each component.

[0082] Example 2:

[0083] This embodiment verifies the temporal functional mechanism of the scaffold at the cellular and molecular levels and evaluates its in vivo repair effect.

[0084] (1) DUSP5-mediated cell rescue and anti-apoptotic mechanisms;

[0085] like Figure 3 and Figure 6 As shown, the protective effect of scaffolds on cells was analyzed in a simulated inflammatory microenvironment. Figure 3 Image A shows the in vitro qualitative immunofluorescence staining images of the expression of pro-inflammatory factors TNF-α, IL-1β and molecular switch DUSP5 in cells. Inflammatory stimulation inhibits DUSP5 expression and upregulates pro-inflammatory factors. SHLP scaffold intervention can restore DUSP5 expression and inhibit the expression of TNF-α and IL-1β.

[0086] Figure 3 Image B shows the in vitro in situ fluorescence detection images of apoptosis cascade proteins Caspase-3 and Bax, and TUNEL-positive apoptotic cells. After intervention, the positive signals of Caspase-3, Bax, and TUNEL were significantly reduced.

[0087] Figure 3 Image C shows the in vivo in situ immunohistochemical verification image of the expression of apoptosis proteins Bax and Caspase-3 at the rotator cuff regeneration termination point in rats 8 weeks after surgery, indicating that the fully functional scaffold can inhibit apoptosis signaling pathways in vivo for a long time. Figure 3 The middle section (D) shows the reconstructed transcriptome heatmap of the expression of key components of the MAPK pathway and apoptosis-related genes in the inflammation model group and the scaffold intervention group, revealing a comprehensive downregulation of MAPK pathway driver genes and pro-apoptotic transcription factors.

[0088] Figure 3 E to H represent quantitative statistical graphs of the relative fluorescence intensity of DUSP5, Bax, Caspase-3 expression levels and the proportion of TUNEL-positive cells, respectively. Figure 3 I and Figure 3 The graphs J in the middle are statistical graphs of the relative quantitative detection of Bax and Caspase-3 at the mRNA transcription level by qPCR, which verify the downregulation of apoptosis genes at the transcription level. Figure 6 The transcriptome heatmap of key gene cluster A, which regulates the reversal of the cellular microenvironment from chronic inflammatory stress to developmental regeneration, shows that inflammation and stress-related genes are suppressed while development-related genes are activated. Figure 6 Image B shows the protein immunoblotting electrophoresis verification of the relative expression levels of intracellular apoptosis marker proteins Caspase-3 and Bax. Figure 6 C and D are the relative quantitative histograms of Western blotting absorbance for Bax and Caspase-3, respectively.

[0089] Figure 6E is a scatter plot of gene ontology enrichment of differentially expressed genes in endoplasmic reticulum stress-related functional items after intervention with a fully functional scaffold. F is a scatter plot of gene ontology enrichment of differentially expressed genes in cartilage / tissue development-related functional items. The results show that differentially expressed genes are significantly enriched in endoplasmic reticulum stress-related functional items and tissue development-related functional items.

[0090] Figure 6 The volcano plot shows the total number of differentially expressed genes with global reversal characteristics between the inflammation model group and the stent intervention group. Figure 6 In the middle H, a two-dimensional scattering map of the proportion of late apoptotic cells in each group was quantitatively detected by Annexin V / PI double staining flow cytometry. The proportion of late apoptotic cells in the SHLP group was significantly reduced, achieving damage repair and protection of the cell population.

[0091] (2) Microenvironment regulation and angiogenesis promotion

[0092] like Figure 4 and Figure 15 As shown, the stent enables the regulation of the pathological microenvironment. Figure 4 Image A shows a global transcriptomic heatmap illustrating the extensive remodeling of gene expression profiles related to endoplasmic reticulum stress, angiogenesis, and inflammatory pathways after scaffold intervention; Image B shows representative fluorescence images from in vitro tubing experiments inducing human umbilical vein endothelial cells to form a lumen network under inflammatory microenvironment, demonstrating that SHLP extract effectively restores the lumen-forming ability of endothelial cells.

[0093] Figure 4 Image C shows the in vitro qualitative immunofluorescence images of the expression and nuclear translocation levels of endoplasmic reticulum stress markers CHOP and GRP78 proteins. The nuclear translocation and expression of CHOP and GRP78 were significantly suppressed in the SHLP group.

[0094] Figure 4 The image in D is an in vitro qualitative immunofluorescence image of the immunoregulatory effect of the transformation of macrophages / progenitor cells from a pro-inflammatory phenotype to an anti-inflammatory phenotype, showing the transformation of cells from an IL-1β and TNF-α mediated pro-inflammatory state to an IL-10 mediated anti-inflammatory state.

[0095] Figure 4 In the middle, E to G are quantitative bar charts of the relative fluorescence intensity of pro-inflammatory factors IL-1β, TNF-α and anti-inflammatory factor IL-10, respectively.

[0096] Figure 4 H and I are scatter plots showing the quantitative analysis of the expression levels of Ang II and VEGFA, pro-angiogenic factors released by rescued cells, respectively. The SHLP group showed the highest expression levels of both factors and the best pro-angiogenic effect.

[0097] Figure 4J and K are quantitative statistical graphs of the relative expression levels of CHOP and GRP78 proteins, respectively. Scaffold intervention significantly reduced endoplasmic reticulum stress damage. Figure 15 This is an in vitro qualitative immunofluorescence assessment image of angiogenesis activation.

[0098] (3) Dilineage differentiation guidance and anabolism

[0099] like Figure 5 , Figure 13 , Figure 14 and Figure 17 As shown, the ability to reconstruct the regional structure of the interface is verified. Figure 5 Image A shows an in vitro dual-lineage differentiation-guided qualitative immunofluorescence image of the expression of type I collagen (a tendon marker) and type II collagen (a cartilage marker) in tendon-bone insertion cells. The image shows that the rescued cells are polarized along the fiber axis and co-express both types of collagen.

[0100] Figure 5 The B-mode is a transcriptome heatmap showing the expression of key differentially expressed genes in the specifically activated myotenoid-associated SCX pathway and chondrogenic-associated Cd44 receptor network. Figure 5 Image C shows an in vivo in situ immunohistochemical staining image and a magnified view of the deposition of type I and type II collagen matrix in the in situ partitioned regenerated tissue at the rotator cuff insertion interface of rats 8 weeks after surgery. It shows the formation of a dense tendinous side dominated by type I collagen and a cartilage transition zone characterized by type II collagen.

[0101] Figure 5 The curve in Figure D shows the quantitative comparison of immunohistochemical integrated optical density of the secretory levels of type I and type II collagen in vivo, with the SHLP group showing the highest secretory level.

[0102] Figure 5 In the middle, E and F are quantitative statistical graphs of fluorescence intensity at the levels of type I and type II collagen secreted in vitro by the rescued cells, respectively, and the protein synthesis level is better than that of the healthy control group.

[0103] Figure 5 China G and Figure 5 The graphs H in the middle represent the statistical results of qPCR quantitative detection of type I and type II collagen expression at the mRNA transcription level, confirming enhanced anabolism.

[0104] Figure 13 This is a long-term evaluation map of cell lineage-specific differentiation in an inflammatory microenvironment, showing long-term qualitative immunofluorescence staining images of cartilage marker SOX9, aggregated proteoglycans, and tendon markers SCX and TNMD under the stimulation of an inflammatory microenvironment in each group.

[0105] Figure 14This is a multidimensional quantitative validation diagram of early dual-lineage transcription factors. In the diagram, A and B are violin plots showing the panoramic distribution of fluorescence intensity of single-cell expression of SOX9 and SCX, respectively; C and D are fine relative quantitative statistical bar charts showing the overall expression levels of SOX9 and SCX in validating the rebound effect of anabolic metabolism.

[0106] Figure 17 This image shows the evaluation of dual lineage differentiation and matrix deposition at the rotator cuff regeneration interface in rats at the early stage 8 weeks post-surgery. It displays representative in situ immunohistochemical microstains and high-magnification local magnification images of type I collagen with tendon-like characteristics and type II collagen secretion and deposition in the fibrocartilage transition zone at the stop point in the early stage 8 weeks after rotator cuff repair surgery in rats.

[0107] (4) In vivo functional repair and biosafety

[0108] like Figure 7 , Figure 16 , Figures 18 to 25 As shown, this study evaluates the functional reconstruction and safety of tendon-bone insertion points in vivo. Figure 7 Image A shows the engineering principle diagram and in-situ photograph of the biomechanical tensile testing device for rat rotator cuff repair; Image B shows the relative quantitative statistical graph of the in vivo biomechanical properties of the ultimate fracture load and structural stiffness after rotator cuff repair. The failure load and stiffness of the SHLP group were the best among all groups.

[0109] Figure 7 The image in C1 shows a panoramic view of the local tissue swelling reduction in rats at 8 and 12 weeks post-surgery, based on T2-weighted sequences, using non-invasive magnetic resonance imaging. The SHLP group showed a uniform low signal characteristic, indicating the resolution of inflammation and edema.

[0110] Figure 7 D shows a bar chart comparing the signal-to-noise ratio of the local MRI signal characteristics at the rotator cuff insertion point at 8 and 12 weeks post-surgery; E shows an in situ immunohistochemical staining image and a high-magnification local magnification of the newly formed, abundant microvascular network supported by low expression of pro-inflammatory factor IL-6 and VEGF in the regeneration microenvironment at the rotator cuff insertion point of rats at 8 weeks post-surgery; F shows the in situ immunohistochemical integrated optical density quantitative statistical curve of the relative expression levels of VEGF and IL-6 in the local tissue at the repaired insertion point.

[0111] Figure 16 This is an in situ immunohistochemical analysis of the regenerated microenvironment 8 weeks after surgery. The figure shows representative in situ immunohistochemical microscopic images and magnified views of the in vivo expression characteristics of endogenous chronic inflammatory marker IL-6 and microvascular construction marker VEGF in regenerated tissue sections of each repair group at 8 weeks after surgery, further demonstrating low IL-6 expression and abundant microvessels.

[0112] Figure 18and Figure 19 Immunohistochemical analysis of the expression of apoptosis proteins Bax and Caspase-3 at the regeneration termination point 8 weeks post-surgery, respectively. The SHLP group suppressed the expression of both proteins to extremely low levels.

[0113] Figure 20 and Figure 21 The images show H&E staining assessments of tendon-bone interface morphology reconstruction at 8 and 12 weeks post-surgery. At 12 weeks post-surgery, the newly formed tendon tissue in the SHLP group was tightly and smoothly integrated with the host bone interface.

[0114] Figure 22 and Figure 23 Masson staining assessment images of interfacial collagen matrix deposition at 8 and 12 weeks post-surgery show that the collagen matrix deposition in the SHLP group is dense and highly parallel and orderly arranged along the stress direction.

[0115] Figure 24 and Figure 25 The images show SO-FG staining assessments of interfacial fibrocartilage matrix deposition at 8 and 12 weeks post-surgery. The SHLP group specifically induced extensive proteoglycan matrix deposition in the tendon-bone transition zone, forming a mature fibrocartilage transition zone. The in vitro cell compatibility and in vivo systemic biocompatibility of the scaffold are assessed in [reference needed]. Figures 9 to 12 As shown. Figure 9 This is a live / dead staining assessment diagram for the in vitro basic cell compatibility of the scaffold. It shows representative qualitative fluorescence images of live / dead cells on days 1, 3, and 7 of rat tendon bone insertion progenitor cells cultured on the surface of the healthy control group, pure SF group, SH group, SHL group, and SHLP group. Green fluorescence represents live cells, and red fluorescence represents dead cells.

[0116] Figure 10 The graphs A through C show the quantitative statistics of the relative survival rate of CCK-8 cells on days 1, 3, and 7 of culture, respectively. Figure 12 This is a graph showing the quantitative analysis of cell mortality rates in different groups under inflammatory stress. Figure 12 Figures A through C show finely quantified statistical bar charts of cell mortality based on the fluorescence ratio of propidium iodide to calcein on days 1, 3, and 7 of cell attachment and culture on the scaffold surfaces of each group. No abnormalities were observed in viable / dead staining, CCK-8 proliferation assays, or cell mortality statistics for any group, indicating that the scaffolds have good in vitro cell compatibility.

[0117] Figure 11 The images show the H&E staining of various organs for in vivo systemic biosafety evaluation. The images present a panoramic view of the histological H&E in situ staining of thin-layer sections of the heart, liver, spleen, lung, and kidney of rats 4 weeks after implantation of various groups of scaffolds. No abnormalities were found in the structure of the major organs, confirming the systemic safety of the system.

[0118] It should be noted that those skilled in the art can make several improvements and modifications without departing from the technical principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.

Claims

1. A pH-responsive tendon-bone interface regenerative coaxial scaffold, characterized in that, The support includes: The outer shell is composed of silk fibroin with a uniaxial orientation; the inner core is composed of hyaluronic acid hydrogel loaded with von Willebrand factor, and the outer shell and the inner core form a coaxial scaffold. A surface modification layer comprising liposomes loaded with Peptide 5, covalently anchored to the surface of the outer shell layer via Schiff base bonds.

2. The pH-responsive tendon-bone interface regenerative coaxial scaffold according to claim 1, characterized in that, The sequence of Peptide 5 is VDCFLSRPTEKT.

3. The pH-responsive tendon-bone interface regenerative coaxial scaffold according to claim 1, characterized in that, The Schiff base bond breaks under acidic conditions with a pH not greater than 5.5, causing the liposomes loaded with Peptide 5 to dissociate and release Peptide 5.

4. The pH-responsive tendon-bone interface regenerative coaxial scaffold according to claim 1, characterized in that, The outer shell is a nanofiber structure, wherein the fiber diameter is 340 nm and the ultimate tensile strength is 12 MPa.

5. A method for preparing the pH-responsive tendon-bone interface regenerative coaxial scaffold according to any one of claims 1 to 4, characterized in that, Includes the following steps: Silk fibroin was dissolved in hexafluoroisopropanol to prepare a silk fibroin solution; Hyaluronic acid and von Willebrand factor are dissolved in water to prepare a mixed solution containing hyaluronic acid and von Willebrand factor; The silk fibroin solution and the mixed solution were electrospun through a coaxial needle, and the coaxial fibers with orientation were collected on a rotating drum receiver. The coaxial core-shell fiber scaffold was obtained by vacuum drying. Liposomes loaded with Peptide 5 and having amino groups on their surface were prepared. The coaxial core-shell fiber scaffold was immersed in a reaction buffer containing the liposomes to obtain the pH-responsive tendon-bone interface regeneration coaxial scaffold.

6. The preparation method according to claim 5, characterized in that, The electrospinning process parameters are: applied voltage 18 kV, receiving distance 15 cm, and roller speed 200 rpm.

7. The preparation method according to claim 5, characterized in that, The preparation of liposomes loaded with Peptide 5 and having amino groups on their surface includes: preparing liposomes loaded with Peptide 5 using a thin-film hydration method, and modifying them with octadecylamine by amino grouping.

8. A biomaterial composition, characterized in that, The biomaterial composition comprises a pH-responsive tendon-bone interface regenerative coaxial scaffold as described in any one of claims 1 to 4.

9. The use of the pH-responsive tendon-bone interface regeneration coaxial scaffold according to any one of claims 1 to 4 in the preparation of a medical device for treating tendon-bone interface defects.

10. The application according to claim 9, characterized in that, The tendon-bone interface defect includes rotator cuff tears.

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