Cysteine-terminal HSPG ligand, scaffold material and application of cysteine-terminal HSPG ligand and scaffold material

Abstract

The invention provides a cysteine-terminated HSPG (heparan sulfate proteoglycan) ligand, a scaffold material and application of the cysteine-terminated HSPG ligand and the scaffold material, the ligand is a polypeptide of CKRSRFFYpIKLIC, the ALP-regulated cysteine terminal is innovatively combined with an HSPG binding motif, and while proteoglycan is combined to mediate mechanical conduction, controllable release and continuous mineralization of phosphate are realized. The fiber structure of the scaffold simulates a collagen network, and promotes ordered deposition of hydroxyapatite generated in a collagen fiber network similar to natural bones, so that an organic-inorganic mutually-enhanced composite structure is formed. According to the invention, the key challenge of biological activity and mineralization regulation and control balance in stent design is solved, and a new normal form is established for constructing an adaptive cell commanding matrix.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a cysteine-terminated HSPG ligand, a scaffold material and applications thereof. Background Art

[0002] Traditional osteoinductive strategies rely on biochemical factors such as bone morphogenetic proteins (BMPs), but these approaches face numerous challenges, including uncontrolled differentiation, ectopic bone formation, and transient efficacy due to rapid degradation. To overcome these limitations, scaffolds that mimic the extracellular matrix (ECM) have emerged as a biomimetic alternative, regulating stem cell fate through cell-ECM interactions. These scaffolds recapitulate the biochemical and structural characteristics of the native ECM, providing sustained mechanical and biochemical signals to promote osteogenesis while avoiding the risks associated with exogenous growth factors. Among various ECM-mimicking strategies, peptide self-assembling scaffolds offer unique advantages due to their molecular precision and tunable self-assembly properties. By incorporating bioactive motifs, these scaffolds can bind to key cellular receptors such as integrins and proteoglycans, thereby enhancing cell adhesion, migration, and differentiation. Furthermore, the modular nature of peptide self-assembling scaffolds allows for the integration of multiple functional domains within a single structure, forming dynamic biomolecular gradients and constructing complex signaling microenvironments. Peptide self-assembly is reversible and adaptable due to non-covalent interactions (such as π-π stacking and hydrogen bonding), enabling scaffolds to respond to environmental cues and support ECM remodeling. These properties make peptide-assembled ECM-mimicking scaffolds a promising strategy for guiding osteogenesis in a more controlled and physiologically relevant manner. A key challenge in the design of osteoinductive scaffolds is constructing composite structures that mimic the collagen fibers in native bone to guide the deposition of hydroxyapatite nanocrystals. Effective biomimetic mineralization systems require a delicate balance between bioactivity and controllable mineralization, ensuring precise spatiotemporal regulation of osteoinductive signals. Enzyme-responsive peptide self-assembly offers a viable solution by coupling mineralization dynamics with cellular activity, particularly the local phosphate supply mediated by alkaline phosphatase (ALP) during osteogenic differentiation. ALP is upregulated during osteogenic differentiation, catalyzing the hydrolysis of phosphate esters and providing high concentrations of inorganic phosphate ions in the local microenvironment, a key step required for physiological bone mineralization (formation of carbon-doped hydroxyapatite (HA) nanocrystals). However, excessive, premature, or uneven phosphate release may lead to heterogeneous mineralization, compromising the integrity and bioactivity of the scaffold.

[0003] Although existing ECM-mimicking scaffolds can promote cell adhesion through integrin binding, they have the following core limitations: (1) The mineralization process usually relies on exogenous calcium and phosphate solution perfusion, which has poor temporal and spatial control and lacks physiological relevance (in vivo mineralization is a local process dominated by cells); (2) The lack of a dynamic response mechanism to the key enzyme for osteogenic differentiation (ALP) leads to the loss of temporal or spatial control of local phosphate concentration, which in turn causes premature mineralization or insufficient mineralization; (3) Scaffolds usually only integrate a single type of cell adhesion signal (such as integrin binding motifs) and fail to integrate and utilize key HSPG binding signals, limiting their ability to simulate the complex signaling environment of natural bone ECM.

[0004] Therefore, it is necessary to develop a novel enzyme-responsive self-assembling scaffold material that can: (a) respond to the upregulated ALP activity during osteoblast differentiation to achieve in situ, on-demand release of phosphate ions, thereby dynamically regulating the mineralization process (overcoming limitations 1 and 2); (b) integrate bioactive motifs that bind to target proteins other than integrins into the molecular design to activate key signaling pathways (overcoming limitation 3); and ultimately achieve the formation of a biomimetic, cell-guided mineralization composite structure within the scaffold. Summary of the Invention

[0005] The present invention aims to provide a cysteine-terminated HSPG ligand, a scaffold material and its application, which achieves spatiotemporal regulation of mineralization dynamics and osteogenesis signals through cysteine-mediated enzyme activity regulation and HSPG targeted binding, and has good scaffold integrity and biological activity.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect of the present invention, a cysteine-terminated HSPG ligand is provided. The ligand is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, comprising the following sequentially linked modules: (a) an N-terminal cysteine; (b) an HSPG binding motif KRSR; (c) an amphiphilic self-assembly domain FFYIKLI; (d) a phosphorylated tyrosine; and (e) a C-terminal cysteine. This polypeptide sequence assembles on the surface of stem cells to form a biomimetic extracellular matrix nanomaterial, dynamically regulating ALP activity and guiding and promoting the directional osteogenic differentiation of hepatocytes. In a second aspect of the present invention, there is provided the use of the polypeptide in preparing a biomimetic mineralized composite osteogenic scaffold material for bone repair.

[0007] In a third aspect of the present invention, a method for preparing a biomimetic mineralized osteogenic scaffold material is provided, the method comprising the following steps: The polypeptide is dissolved in a culture medium and then mixed with a suspension of human bone marrow mesenchymal stem cells for incubation. Under the guidance of the endogenous HSPG and ALP of the stem cells, a nanofiber structure is formed, and an extracellular matrix (ECM) biomimetic scaffold material is formed on the cell surface.

[0008] Furthermore, the phosphorylated tyrosine in the polypeptide is dephosphorylated under the catalysis of the endogenous ALP of the stem cell, releasing inorganic phosphate and driving the peptide segments to self-assemble into a nanofiber structure.

[0009] Furthermore, the concentration of the polypeptide dissolved in the culture medium is ≥0.2 mM.

[0010] Furthermore, the culture medium is an osteogenic induction medium, and optionally, OriCell® culture medium containing 10% FBS can be used.

[0011] Furthermore, the incubation conditions include: a temperature of 36° C.-38° C. and a time of ≥72 hours.

[0012] In a fourth aspect of the present invention, a biomimetic mineralized composite osteogenic scaffold material prepared by the method is provided.

[0013] In a fifth aspect of the present invention, there is provided the use of the scaffold material in preparing a bone defect repair implant.

[0014] Furthermore, the scaffold material guides and promotes osteogenic differentiation.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. The present invention provides a cysteine-terminated HSPG ligand, which is a cysteine-terminated HSPG ligand containing a phosphate amino acid group. It can not only maintain the binding activity of HSPG, but also endow the peptide segment with responsiveness to alkaline phosphatase (ALP) by introducing phosphorylated tyrosine, thereby triggering advanced self-assembly and mineralization at a specific cell differentiation stage (high ALP expression), and has good spatiotemporal control characteristics. Compared with the mineralization process in traditional materials without a specific response mechanism, this design can achieve adaptive mineralization dependent on the cell state, improving the accuracy and physiological relevance of osteogenic induction; at the same time, it retains the binding function of HSPG and ensures basic biological functions such as cell adhesion and signal transduction.

[0016] 2. The present invention provides a cysteine-terminated HSPG ligand that introduces the amphiphilic sequence FFYIKLI to promote self-assembly. The guiding peptide segment spontaneously forms a nanofiber structure through π-π stacking and hydrogen bond interactions, simulating the collagen fiber matrix in natural bone tissue. Compared with non-self-assembling molecules, this ordered structure is more conducive to calcium and phosphate deposition, cell adhesion and signal transduction, and constructs a scaffold that is closer to the native ECM (extracellular matrix) structure. It achieves efficient self-assembly under mild conditions without the need for exogenous inducing factors.

[0017] 3. Introduction of cysteine ​​residues at both ends: On the one hand, zinc chelation is used to transiently inhibit ALP activity, regulate the dephosphorylation process, achieve controllable phosphate release, and avoid premature calcification; on the other hand, it simulates the osteogenic effect of N-acetylcysteine ​​(NAC) to enhance osteoblast differentiation and mineralization. This dual-functional strategy integrates enzyme activity regulation and osteogenesis promotion in a single molecule, which is innovative in existing technologies.

[0018] 4. In the present invention, ALP triggers self-assembly and synchronous mineralization mechanism: peptide segments are dephosphorylated under the action of ALP, accompanied by enhanced structural order, achieving a synergistic response of structure and function; the synchronously released phosphate promotes in situ calcium-phosphate deposition, which is embedded in the peptide scaffold fibers to form a biomimetic mineralized structure; compared with the existing multi-step mineralization process, this method can complete assembly and mineralization in one step, simplifying the process and improving biocompatibility.

[0019] 5. The scaffold structure induces extracellular calcification and promotes the expression of osteogenic genes: the scaffold forms a stable fiber network on the cell surface, enhances cell adhesion and promotes local calcification; significantly increases the expression of osteogenic-related genes (such as Runx2 、 Col1a1 、 Ocn It can effectively regulate the expression of osteogenic factors (such as BMP) and proteins, and has a higher osteogenic induction efficiency than traditional bone differentiation induction medium; it shows the potential application prospect of replacing traditional osteogenic induction factors (such as BMP) and avoiding their potential side effects and instability.

[0020] 6. The scaffold structure promotes focal adhesion maturation and actin contraction: The peptide scaffold induces hMSCs to form large and mature focal adhesions, enhancing cytoskeletal stability; significantly increases pMLC expression, suggesting increased intracellular tension, which is conducive to the activation of mechanical transduction-mediated differentiation pathways; and helps to build a microenvironment with biomechanical activation function, which is an advantage that traditional rigid or inert materials cannot match.

[0021] 7. YAP-mediated activation of downstream mechanical signaling pathways: The scaffold can promote the translocation of YAP from the cytoplasm to the nucleus in a short period of time, indicating that it can rapidly activate the cell's mechanosensitive transcriptional program; the YAP activation level is correlated with the peptide concentration, proving that the scaffold can regulate stem cell fate decisions in a dose-dependent manner; it realizes a full-link regulatory mechanism from the material-cell interface to gene expression, which is extremely innovative and practical in the design of tissue engineering scaffolds. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Enzyme-responsive peptides self-assemble into nanofibrous scaffolds. Figure 1 a. Design the chemical structure of the peptide. Figure 1 b. TEM images of the two peptides in PBS buffer (1 mM) before and after ALP treatment (5 U). Figure 1 c. Circular dichroism (CD) spectra of the two peptides in PBS buffer (200 μM) before and after ALP treatment (5 U). Figure 1 d. Dephosphorylation kinetics of the two peptides under ALP (5 U) treatment (37°C).

[0024] Figure 2 In vitro assembly and osteogenic induction of enzyme-responsive peptides. Figure 2 a. Cell viability of hMSCs after treatment with different concentrations of peptides for 3 days. Figure 2 b. Confocal fluorescence images of hMSCs after 3 days of peptide treatment (F-actin in green, DAPI in blue, and Congo Red showing peptide assemblies in red). Figure 2 c. SEM images of hMSCs cultured under different treatment conditions for 3 days (yellow arrows indicate fiber scaffolds). Figure 2 d. Alizarin Red staining showed calcium deposition after 14 days.

[0025] Figure 3 Scaffold mineralization and osteogenesis gene / protein expression Figure 3 a. SEM and EDS elemental distribution images (calcium deposition) of hMSCs treated with CKRSRFFYpIKLIC for 14 days. Figure 3 b. Expression levels of osteogenic genes (Runx2, Ocn, Spp1, Colla1) in hMSCs under different treatments. Figure 3 cd. Western blot results and quantitative analysis of osteoblast marker proteins (RUNX2, COL1A1, OPN, SP7).

[0026] Figure 4 Enhances focal adhesion maturation and cell traction for scaffolds. Figure 4 a. Distribution of Paxillin (focal adhesion marker protein) in hMSCs after 3-day treatment with different concentrations of CKRSRFFYpIKLIC. Figure 4 b. pMLC fluorescence images and quantitative analysis of the pMLC / Actin intensity ratio (reflecting cell traction force).

[0027] Figure 5 For YAP nuclear translocation analysis. Figure 5 a. Nuclear-cytoplasmic distribution of YAP after treatment with different concentrations of peptides for 4 h (green: YAP, blue: DAPI). Figure 5 b. Quantification of YAP nuclear-cytoplasmic fluorescence intensity ratio (violin plot).

[0028] Figure 6 This is the LC-MS spectrum of CKRSRFFYpIKLIC.

[0029] Figure 7 This is the LC-MS spectrum of KRSRFFYpIKLI. Figure 8 The biocompatibility of the peptides was verified by live / dead staining of cells.

[0030] Figure 9 Metallographic microscopy images of hydroxyapatite nanocrystals in the extracellular scaffold formed in hMSCs after 21 days of treatment with 200 μM peptide. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0032] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, 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 invention belongs. In the event of any conflict, the present specification shall take precedence.

[0033] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0034] In order to solve the technical problems of the present invention, the overall concept of the present invention is as follows: As a typical embodiment, the present invention provides a cysteine-terminated HSPG ligand, wherein the ligand is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, and the polypeptide integrates the following functions: (1) Enzyme-responsive molecular assembly and mineralization: Tyrosine phosphorylation modification achieves ALP-triggered dephosphorylation, drives molecular assembly and local phosphate release, and promotes calcium phosphate deposition attached to molecular assembly fibers.

[0035] (2) Regulation of ALP activity: Cysteine ​​temporarily inhibits ALP by chelating zinc ions, preventing premature mineralization caused by excessive local phosphate enrichment.

[0036] (3) HSPG targeting: The FF-modified KRSR motif selectively binds to transmembrane HSPG and enhances cell adhesion by forming a molecularly assembled extracellular scaffold, guiding and promoting the osteogenic differentiation of stem cells.

[0037] (4) Self-assembly ability: The amphiphilic peptide segment of FFYpIKLI drives the formation of a fiber network through π-π stacking and hydrogen bonding. After dephosphorylation by ALP, the hydrophobicity of FFYIKLI is relatively increased, further promoting the assembly to form a stable fiber network.

[0038] As a typical embodiment, the present invention provides an organic-inorganic composite nano-osteogenic scaffold material functionalized with heparin sulfate proteoglycan (HSPG) ligands that can be self-assembled in response to alkaline phosphatase (ALP).

[0039] This material has good biocompatibility, mineralization controllability and osteogenic induction ability, and has broad practical value and clinical transformation potential.

[0040] (1) Tissue engineering scaffold materials: The functionalized polypeptide of the present invention can form a mineralized fibrous scaffold in situ on the cell surface through specific binding to proteoglycans on the surface of mesenchymal stem cells (MSCs) and its enzyme-responsive self-assembly mechanism, simulating the structure and function of natural bone matrix, and is suitable for: (2) Scaffold construction in bone tissue engineering; as a cell carrier material in bone defect repair, especially suitable for local implantation of small defects; 3D printing bio-ink additive to enhance the bioactivity and mineralization capacity of printed structures.

[0041] (3) Orthopedic clinical treatment and regenerative medicine applications: The intelligent responsive material provided by the present invention does not need to rely on exogenous osteogenic factors such as BMPs, thus avoiding their side effects and cost issues. It is particularly suitable for: minimally invasive bone repair treatment containing stem cell injection; auxiliary filling materials in vertebral fusion or fracture fixation to promote bone fusion; microstructural reconstruction of osteoporosis patients to improve local mineralization levels.

[0042] (4) Drug delivery and intelligent nanostructure construction: The self-assembly characteristics can be extended to construct other functional nanostructures for: Enzyme-responsive drug delivery systems: By modifying the ligand sequence, materials that respond to other enzymes (such as MMP, Cathepsin K) can be developed; combined with bone-forming drugs and anti-inflammatory drugs to construct a multifunctional bone repair system and achieve combined treatment.

[0043] (5) Bionic materials and mechanism research platform: This material is a platform that integrates structural bionics, signal targeting and mechanical response. It is suitable for: research on stem cell fate regulation mechanisms; in vitro models of bone development and mineralization mechanisms; modular bionic assembly templates in cross-research of synthetic biology and protein engineering.

[0044] In summary, the present invention not only has a highly feasible transformation prospect in existing bone tissue engineering and stem cell research, but also provides important technical support and theoretical basis for the future development of intelligent bionic material systems.

[0045] The present application will be described in detail below with reference to examples and experimental data.

[0046] Example 1. Design of HSPG ligands for enzyme-responsive assembly 1. As shown in Scheme 1, we designed a cysteine-terminal HSPG binding site containing a phosphate amino acid. The HSPG-binding motif KRSR was selected as the functional domain that binds to heparan sulfate proteoglycans and serves as the core structural unit.

[0047] To confer self-assembly capability, KRSR was conjugated with the amphiphilic peptide FFYIKLI, which promotes intermolecular interactions through π-π stacking and hydrogen bonding ( Figure 1 a). Phosphorylation of tyrosine (Y) residues confers enzymatic responsiveness to the peptide, enabling ALP-triggered dephosphorylation to drive peptide assembly and phosphate release. The introduction of cysteine ​​has a dual role: first, it transiently inhibits ALP activity by chelating zinc ions, providing a regulatory mechanism for controlled phosphate release; second, its structure mimics the osteogenic effects of N-acetylcysteine ​​(NAC), which accelerates osteoblast maturation and mineralization.

[0048] To preserve dual functionality, we introduced cysteines at both ends of the peptide chain, resulting in the sequence CKRSRFFYpIKLIC. As a control, we synthesized the peptide KRSRFFYpIKLI, which lacks the terminal cysteines, to distinguish between cysteine-mediated ALP regulation and the independent effects of NAC-like osteogenesis enhancement.

[0049] 2. Chemical synthesis of peptides (1) Design of two enzyme-responsive peptides: Peptide in the experimental group: CKRSRFFYpIKLIC (containing cysteine ​​at the C-terminus and N-terminus, and phosphorylated tyrosine residues).

[0050] Control peptide: KRSRFFYpIKLI (no cysteine ​​modification).

[0051] (2) Synthesis method: The product was synthesized by solid phase synthesis with a purity of ≥95%. The product was purified by HPLC (using a C18 column and a mobile phase consisting of 0.1% TFA in acetonitrile / water, gradient elution), and the purity and molecular weight were verified by LC-MS.

[0052] 3. Characterization of self-assembly behavior (1) Transmission electron microscopy (TEM) analysis: The peptide was dissolved in PBS buffer (1 mM, pH 7.4) and added dropwise to the carbon film copper grid. uranyl acetate) and then observed.

[0053] The results are as follows Figure 1 (b) Both CKRSRFFYpIKLIC and KRSRFFYpIKLI self-assemble into nanofiber networks at a concentration of 1 mM. Notably, the nanofibers formed by CKRSRFFYpIKLIC containing a terminal cysteine ​​are thinner and more delicate, indicating that cysteine ​​termination influences the nanofiber morphology.

[0054] (2) Circular dichroism (CD): Analyzed at a wavelength of 200-250 nm (Chirascan V100 spectrometer, 1 mm pathlength).

[0055] The results are as follows Figure 1 c: Before ALP treatment, both peptides showed characteristic random coil peaks (negative peak at 200 nm, positive peak at 220 nm). After ALP treatment (5 U, 37°C), the signal intensity increased, indicating an increase in the proportion of β-sheet structure.

[0056] After ALP treatment, both peptides formed denser networks while retaining their basic fibrillar structure. This morphological transition was accompanied by an increase in CD signal intensity, but the peak shape and wavelength did not change significantly, indicating enhanced supramolecular order without altering the secondary structure.

[0057] 4. Enzyme responsiveness kinetics (1) ALP-mediated dephosphorylation analysis: The peptide solution (200 μM) was incubated with ALP (5 U) (37°C), and samples were taken at regular intervals to detect the dephosphorylation rate by HPLC.

[0058] The results of ALP-mediated dephosphorylation kinetics analysis are shown in Figure 2. Figure 1 As shown in d: The peptide CKRSRFFYpIKLIC can significantly delay the release of phosphate. The terminal cysteine ​​in CKRSRFFYpIKLIC significantly delays the enzymatic cleavage of the phosphate group, confirming its role in regulating ALP activity.

[0059] Example 2: Preparation and in vitro mineralization of biomimetic mineralized composite scaffolds 1. Enzyme-triggered assembly to form extracellular scaffolds (1) Dissolve CKRSRFFYpIKLIC (200 μM) in osteogenic induction medium (OriCell® medium containing 10% FBS) containing hMSCs.

[0060] (2) ALP (5 U) was added and incubated at 37°C for 72 hours to form an extracellular fiber network.

[0061] 2. Characterization: (1) Confocal microscopy The results are as follows Figure 2 (b) Congo Red staining reveals a red, fibrous scaffold tightly surrounding the cells. KRSRFFYpIKLI formed only a small amount of scaffold in cell culture, while CKRSRFFYpIKLIC formed a visible extracellular scaffold on the cell surface. The scaffold fiber density increased with increasing peptide concentration, demonstrating its superior potential for constructing extracellular matrix-mimetic scaffolds directly on the surface of hMSCs.

[0062] (2) Scanning electron microscopy (SEM) The results are as follows Figure 2 c: Scanning electron microscopy (SEM) further verified the above results, showing that a fibrous scaffold was formed on the surface and around hMSCs.

[0063] (3) Calcium deposition results To preliminarily evaluate the osteoinductive potential of the peptide, calcium deposition was detected by Alizarin red staining, and optical images showed ( Figure 2 d) Compared with the KRSRFFYpIKLI-treated group and the standard stem cell osteogenic induction medium, CKRSRFFYpIKLIC significantly enhanced calcium deposition, indicating that it has the ability to effectively promote hMSC osteogenic differentiation. EDS confirmed that layered calcium phosphate crystals were deposited in the scaffold ( Figure 3 a).

[0064] Example 3. Biocompatibility and cytotoxicity of peptides To evaluate the biocompatibility of the designed peptides, we examined the biocompatibility of human bone marrow mesenchymal stem cells after treatment with different concentrations of the peptides. The survival rate of human mesenchymal stem cells (hMSCs).

[0065] 1. CCK-8 assay hMSCs (5 × 10 3 / well) were exposed to peptide solutions (50-500 μM) for 72 hours: result Figure 2As shown in a, KRSRFFYpIKLI had no significant effect on cell viability at all tested concentrations, while CKRSRFFYpIKLIC slightly decreased hMSC cell viability at a concentration of 500 μM.

[0066] 2. Live / Dead dyeing Given that stem cell differentiation often comes at the expense of decreased proliferation capacity, we further assessed potential cytotoxicity using live / dead cell viability assays.

[0067] The results are as follows Figure 8 As shown in the results, the proportion of living cells was >95%, and both peptides were non-toxic to hMSCs, suggesting that the decrease in cell activity observed in the CCK-8 assay was mainly due to peptide-induced osteogenic differentiation of stem cells rather than cytotoxic effects. Example 4: ALP-responsive assembly of CKRSRFFYpIKLIC promotes biomimetic mineralization and hMSCs osteogenic differentiation 1. Mechanism research In native bone tissue, collagen self-assembly precedes mineral deposition, forming a fibrillar matrix that chelates calcium ions and provides nucleation sites for ordered calcium phosphate (CaP) crystals. Inspired by this process, our design recapitulates the temporal interplay between matrix assembly and mineralization.

[0068] like Figure 1 As shown in Figure 3, CKRSRFFYpIKLIC can spontaneously assemble into a fibrous network before enzyme activation, with a structure similar to collagen fibers. Upon ALP-catalyzed dephosphorylation, the peptide locally releases phosphate and promotes in situ CaP crystallization ( Figure 3 a and Figure 9 ) and enhance further assembly of the peptide ( Figure 1 b). This synergistic response enables the embedding of CaP nanocrystals into the fiber peptide scaffold, forming a composite scaffold material that mimics the mineralized matrix.

[0069] SEM analysis ( Figure 3 a) Showing that the scaffold is embedded with layered CaP crystals, resembling the classic layer-by-layer growth of hydroxyapatite from an amorphous precursor. The upregulation of osteogenic markers after CKRSRFFYpIKLIC treatment further validates this biomimetic mineralization process.

[0070] 2. Expression of osteoblastic markers qPCR analysis: Gene expression levels in the CKRSRFFYpIKLIC group (vs. GAPDH) after 21 days of culture. The primers used are shown in the table below.

[0071] Table 1

[0072] The results of gene expression analysis were as follows Figure 3 As shown in b, Runx2 、 Ocn 、 Spp1 and Col1a1 Significant increase.

[0073] Western Blotting results are as follows Figure 3 As shown in c and 3d, the protein levels of RUNX2, COL1A1, OPN, and SP7 increased, and their expression levels exceeded those under traditional osteogenic induction conditions.

[0074] In summary, the dynamic organic-inorganic composite biomimetic extracellular matrix constructed by CKRSRFFYpIKLIC not only mimics the natural bone mineralization process but also provides a potent osteogenic induction signal. The promotion of crystalline CaP deposition within the scaffold highlights its potential to enhance bone regeneration by cultivating a mature, functional mineralized matrix. Example 5: Osteoinductive scaffolds promote hMSCs focal adhesion maturation and actomyosin contractility 1. To clarify how this biomimetic scaffold converts its biochemical and structural signals into cellular responses, we conducted The mechanical response behavior of hMSCs interacting with CKRSRFFYpIKLIC-induced scaffolds was further investigated.

[0075] Given the clear role of focal adhesion dynamics and cytoskeletal tension in regulating stem cell fate, we evaluated the We evaluated whether the osteoinductive microenvironment drives osteogenic differentiation through mechanotransduction signals.

[0076] Immunofluorescence staining results Figure 4 As shown in a, hMSCs in the control group showed small, punctate paxillin (focal adhesion marker protein) was localized to the ends of stress fibers, characteristic of immature or nascent focal adhesions. In contrast, in the CKRSRFFYpIKLIC-treated group, large, elongated focal adhesions were formed as the peptide concentration increased, and these focal adhesions were widely distributed on the ventral surface of the cells, indicating the formation of stable, mature focal adhesion structures.

[0077] 2. To verify whether these structural changes affect cell tension, we used phosphorylated myosin light chain (pMLC) was used as a marker of actomyosin activation for visualization analysis.

[0078] like Figure 4 b, CKRSRFFYpIKLIC treatment resulted in thicker and more pronounced pMLC fibers. F-actin alignment indicates increased cytoskeletal tension.

[0079] Quantitative analysis of the pMLC / actin fluorescence intensity ratio was as follows Figure 4As shown in c, the concentration-dependent increase confirmed that CKRSRFFYpIKLIC treatment could enhance cell traction force.

[0080] In summary, the osteoinductive scaffold not only promotes the maturation of focal adhesions but also enhances intracellular tension, thereby constructing a biomechanical microenvironment that is conducive to mechanical conduction-driven osteogenic differentiation. Example 6: Osteoinductive scaffolds activate YAP-mediated mechanotransduction in hMSCs Based on the observation of focal adhesion maturation and enhanced actomyosin contractility, we further explored this Whether these biomechanical changes are converted into downstream mechanotransduction signals. Yes-associated protein (YAP) is a A classical mechanosensitive transcriptional coactivator that translocates from the cytoplasm to the The YAP subunit is expressed in the nucleus, thereby regulating the gene expression program related to stem cell fate. Cell localization was used as a functional characterization indicator to evaluate the mechanical transduction signal induced by CKRSRFFYpIKLIC in role in hMSCs.

[0081] Nuclear localization results Figure 5 As shown in a, CKRSRFFYpIKLIC treatment for only 4 hours can induce YAP Significant nuclear translocation indicates the activation of early mechanotransduction signaling.

[0082] Quantitative analysis such as Figure 5 As shown in b, the nuclear localization of YAP increased in a peptide concentration-dependent manner, further confirming that the biomechanical signal of the scaffold rapidly activated the YAP pathway.

[0083] These results suggest that osteoinductive scaffolds initiate an integrated set of mechanotransduction mechanisms by coordinating extracellular signals with transcriptional regulation of stem cell fate.

[0084] In summary, the present invention has developed a HSPG ligand with a cysteine ​​terminal, which can be activated by enzyme response. The peptide CKRSRFFYpIKLIC innovatively combines the ALP regulatory cysteine ​​terminal with the HSPG binding motif. Polysaccharide mediates mechanical conduction while achieving controlled release of phosphate to realize extracellular biomimetic scaffold Sustained mineralization. The fibrous structure of the scaffold mimics the collagen network, promoting the deposition of hydroxyapatite in the collagen network similar to natural bone. Cell experiments showed that the scaffold induced and enhanced hMSC osteogenic differentiation by promoting the maturation of focal adhesions and enhancing cell traction, thereby achieving YAP-mediated transcriptional activation, and its efficacy surpassed that of traditional osteogenic induction culture media. This strategy solves the key challenge of balancing bioactivity and mineralization regulation in scaffold design, and establishes a new paradigm for the construction of adaptive cell-directed matrices. Future studies will explore its performance in in vivo bone regeneration and long-term scaffold remodeling, and promote the clinical translation of this strategy into orthopedic treatment.

[0085] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0086] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0087] Obviously, those skilled in the art may make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if such changes and modifications fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are intended to include such changes and modifications.

Claims

1. A cysteine-terminated HSPG ligand, characterized in that: The ligand is a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, comprising the following modules connected in sequence: (a) N-terminal cysteine; (b) HSPG binding motif KRSR; (c) amphiphilic self-assembly domain FFYIKLI; (d) phosphorylated tyrosine; and (e) C-terminal cysteine.

2. Use of the cysteine-terminated HSPG ligand according to claim 1 in the preparation of a biomimetic mineralized composite osteogenic scaffold material for bone repair.

3. A method for preparing a biomimetic mineralized composite osteogenic scaffold material, characterized in that: The method comprises the following steps: The polypeptide according to claim 1 is dissolved in a culture medium, and then mixed with human bone marrow mesenchymal stem cells for incubation and culture, and an extracellular biomimetic mineralized composite osteogenic scaffold material with a nanofiber structure is formed on the cell surface under the guidance of the endogenous HSPG and ALP of the stem cells.

4. The method according to claim 3, characterized in that The phosphorylated tyrosine in the polypeptide is dephosphorylated under the catalysis of the endogenous ALP of the stem cell, releasing inorganic phosphate and driving the peptide segments to self-assemble into a nanofiber structure.

5. The method according to claim 3, wherein The pH of the phosphate buffer is 7.4-8.

0.

6. The method according to claim 3, wherein The concentration of the polypeptide dissolved in the culture medium is ≥0.2 mM.

7. The method according to claim 3, characterized in that The incubation conditions include: a temperature of 36° C.-38° C. and a time of ≥72 hours.

8. A biomimetic mineralized composite osteogenic scaffold material prepared by the method according to any one of claims 3 to 7.

9. Use of the scaffold material according to claim 8 in the preparation of bone defect repair implant products.

10. The use according to claim 9, characterized in that The scaffold material guides and promotes osteogenic differentiation.

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