An evaluation method for hydrogel mesoscopic mechanical homeostasis, amphiphilic peptide hydrogel biological stabilizer and application thereof in preparation of bone regeneration promoting drugs

Abstract

This invention relates to amphiphilic peptide hydrogels and their design and applications, specifically to a method for evaluating the mesoscopic mechanical stability of hydrogels, an amphiphilic peptide hydrogel biostabilizer, and its application in the preparation of bone-promoting drugs. To address the shortcomings of existing technologies where viscoelastic drift around cells is caused by both AGEs load and functional mechanical disturbances, resulting in a lack of stable mechanical background for osteogenic signals and easily leading to jawbone regeneration failure in high-glucose diets, this invention provides a method for evaluating the mesoscopic mechanical stability of hydrogels using DTR=CV. output / CV input Quantifying the degree of dispersion transfer from pathological perturbation input to mesoscopic mechanical output transforms biostabilization from a qualitative description into a calculable, comparable, and cross-materials-verifiable engineering indicator. Simultaneously, it provides an amphiphilic peptide hydrogel biostabilizer as a mesoscopic biostabilizer, maintaining stable viscoelasticity in the pericellular microdomain and accommodating matrix remodeling and mineralization.

Description

Technical Field

[0001] This invention relates to amphiphilic peptide hydrogels and their design and applications, specifically to a method for evaluating the mesoscopic mechanical stability of hydrogels, amphiphilic peptide hydrogel biostabilizers, and their application in the preparation of bone regeneration drugs. Background Technology

[0002] Jaw and alveolar bone defects are a common clinical problem faced by oral and maxillofacial surgery, periodontology, implant restoration, and trauma repair. Patients with diabetes and chronic hyperglycemia often experience microvascular dysfunction, prolonged inflammation, increased risk of infection, bone turnover imbalance, and decreased osteoblast function. Delayed healing, insufficient bone volume, decreased mineralization, and nonunion are common problems in extraction socket healing, implant site bone augmentation, periodontal bone defect repair, and post-traumatic / tumor reconstruction of the jaw. Compared to long bones, the jaw is exposed to a complex oral microbiota, salivary environment, and continuous masticatory load. The defect area not only needs to form sufficient new bone but must also maintain stable bone remodeling and mineralization quality under dynamic functional loads. Therefore, the core clinical challenge of jaw regeneration in hyperglycemia is not simply "insufficient osteogenic signals," but rather the difficulty for osteoblasts and mesenchymal stem cells to receive and integrate osteogenic signals stably and over a long period under continuous perturbation of the pathological microenvironment.

[0003] Currently, treatment strategies for bone defects associated with high glucose or diabetes mainly include autologous / allogeneic bone transplantation, bone regeneration guided by barrier membranes and scaffold materials, delivery of growth factors such as BMP, stem cell or exosome therapy, anti-inflammatory and antioxidant materials, pro-angiogenic materials, and intervention in the AGEs / RAGE pathway. These strategies are valuable in providing osteoconductive scaffolds, enhancing osteogenic induction, and improving local inflammation and vascularization. However, they still have three shortcomings in clinical translation: First, growth factor and cell therapies are significantly affected by the high glucose environment, resulting in short efficacy windows, large batch-to-batch variability, and high costs. Second, most bone regeneration materials focus on bulk porosity, degradation rate, static elastic modulus, or macroscopic rheological properties, making it difficult to reflect the microscale mechanical state actually perceived by cells. Third, existing materials mostly provide osteogenic stimulation or passive support in a one-time manner, lacking the ability to actively maintain the homeostasis of the microenvironment around cells under the continuous accumulation of AGEs and repeated disturbances from chewing loads.

[0004] Advanced glycation end products (AGEs) are a significant pathological factor contributing to jawbone regeneration failure in high-glucose conditions. Under high glucose levels, AGEs are continuously generated and cross-link with long-lived extracellular matrix (ECM) proteins such as collagen, leading to abnormal matrix fiber arrangement, viscoelastic drift, impaired mineralization deposition, and damaged cell-matrix adhesion. Cell fate determination depends on the integration of mechanical information at the 1-50 μm mesoscopic scale surrounding the cell, including adhesion, tension, stress relaxation, viscoelastic dissipation, and new ECM deposition, rather than simply being determined by the overall stiffness of the material. The continuous perturbation caused by high glucose / AGEs alters this mesoscopic mechanical niche around the cell, preventing MSCs from maintaining adequate mechanical input during the early osteogenic initiation window. This, in turn, affects mechanotransduction and osteogenic transcriptional programs such as Integrin-Fak, Hippo / YAP-TAZ, and Runx2, ultimately resulting in insufficient bone formation and decreased mineralization quality.

[0005] In recent years, mechanobiology research has evolved from the understanding that "the static stiffness of materials affects cell fate" to a new stage where "dynamic viscoelasticity, stress relaxation, and cell deposition (ECM) jointly determine cell fate." Materials such as hydrogels, bioactive peptides, and biodegradable scaffolds are beginning to incorporate tunable viscoelasticity, dynamic cross-linking, and remodelable networks. However, existing research largely focuses on mechanical induction under normal physiological conditions, paying less attention to the persistent drift of mesoscopic mechanical baselines in chronic disease environments. Particularly in high-glucose jawbone defects, the superposition of AGEs chemical cross-linking and masticatory mechanical perturbations leads to a continuous temporal and spatial deviation of pericellular mechanical signals from the osteogenic adaptation range. This resulting "loss of mesoscopic mechanical homeostasis" is a key bottleneck that traditional biochemical osteogenic induction struggles to overcome.

[0006] In summary, one of the essential reasons for the failure of jawbone regeneration in high-glucose individuals is the viscoelastic drift around cells caused by the combined effects of AGEs load and functional mechanical disturbances, which results in a lack of stable mechanical background for osteogenic signals. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies, such as viscoelastic drift around cells caused by AGEs load and functional mechanical disturbances, which leads to a lack of stable mechanical background for osteogenic signals and makes high-glucose jawbone regeneration failure. The invention provides a method for evaluating the mesoscopic mechanical stability of hydrogels, an amphiphilic peptide hydrogel biostabilizer, and its application in the preparation of osteogenic drugs.

[0008] The inventive concept of this invention is as follows: This invention proposes for the first time a bone repair treatment paradigm of Micro-Mechanical Homeostasis Reset (MMHR). The goal is not to optimize static body stiffness, but rather to regulate the mesoscopic (cell-to-surround) viscoelastic homeostasis of actual cell integration, and to design a system that incorporates Ca²⁺... + ViscoClamp, a peptide hydrogel with affinity for and AGEs (Advanced Glyceryl Acetate) molecules, utilizes DDDEEKF as a mineralization / calcium-binding module, an AGEs adsorption module, and a cysteine-stabilizing module replacing the AGEs adsorption module's terminal to form an injectable, interpenetrating ECM for MSC deposition. Under high glucose / AGEs perturbation, ViscoClamp locally adsorbs and buffers the AGEs load, compressing highly discrete pathological inputs into low discrete viscoelastic outputs while preserving the cell matrix remodeling, collagen assembly, and mineralization deposition capabilities. This invention transforms biostabilization into an input-output problem, using the dispersion-transfer ratio (DTR=CV). output / CV input As a quantitative indicator, it quantitatively characterizes how variable glycation inputs are compressed into stable mesoscopic mechanical outputs. Simultaneously, combining atomic force microscopy (AFM) mesoscopic mechanical measurements, osteogenic function analysis, omics mechanism analysis, and multi-species models (rat, rabbit, beagle dog, and rhesus monkey), it systematically verifies the mechanism, efficacy, and safety of its promotion of high-glycation jawbone regeneration.

[0009] To achieve the above objectives and inventive concepts, the technical solution provided by this invention is as follows:

[0010] A method for evaluating the mesoscopic mechanical steady state of hydrogels, characterized by the following steps:

[0011] Step 1: Add gradient concentrations of adsorbate to multiple hydrogels of equal volume, mix thoroughly, and let stand for x hours to obtain multiple sets of test samples; x takes the value of 1 to 2.

[0012] Step 2: Measure the apparent elastic modulus E of the sample to be tested. exp The power-law exponent α, gel relaxation time τ, local gel unit stiffness E1, effective viscosity η, and fiber network stiffness E2;

[0013] Step 3, Define CV input The coefficient of variation of the adsorbate concentration is calculated from the standard deviation / mean of the adsorbate concentration described in step 1.

[0014] Define that the output parameter m belongs to {E} expGiven the set of adsorbates at gradient concentrations { , α, τ, E1, η, E2}, calculate the standard deviation / mean of the output parameter m, and obtain the coefficient of variation (CV) of the output parameter m. m Coefficient of variation (CV) m Including E exp The coefficients of variation of α, τ, E1, η, E2;

[0015] Calculate E exp The arithmetic mean of the coefficients of variation of α, τ, E1, η, and E2 is used to obtain the overall dispersion CV. output ;

[0016] Step 4, based on CV output / CV input ×100% yields the Dispersion Transfer Ratio (DTR);

[0017] Step 5: If 4.5%≤DTR≤5%, the hydrogel is considered to have mesoscopic mechanical stability; otherwise, the hydrogel has insufficient mesoscopic mechanical stability.

[0018] Furthermore, it also includes step 6, in which steps 2-5 are repeated for multiple groups of test samples at different time points after x hours of placement in step 1, to obtain the corresponding mesoscopic mechanical stability results, in order to test time robustness.

[0019] Furthermore, step 2 specifically involves:

[0020] Multiple groups of samples were examined using atomic force microscopy, and the creep compliance was obtained by combining it with dynamic creep indentation. Simultaneously, the apparent elastic modulus E was obtained using quasi-static indentation. exp ;

[0021] All creep compliances were analyzed using a self-similar hierarchical model and a power-law model. Creep compliance-time curves within the range of 0.01–0.1s were fitted to obtain the local gel unit stiffness E1, fiber network stiffness E2, and gel relaxation time τ.

[0022] The effective viscosity η is calculated based on the local gel unit stiffness E1 and the gel relaxation time τ.

[0023] The power-law exponent α can be obtained by taking a single logarithm of the creep compliance-time curve and performing linear fitting.

[0024] The present invention also provides an amphiphilic peptide hydrogel biostabilizer, wherein the biostabilizer has the mesoscopic mechanical stability evaluated by the above-mentioned method, and is characterized in that it includes an amino acid sequence; the amino acid sequence includes a calcium-binding module and an AGEs adsorption module connected in sequence, and the terminal alanine A of the AGEs adsorption module is replaced with cysteine ​​C.

[0025] Furthermore, the calcium-binding module is DDDEEKF;

[0026] The AGEs adsorption module is (RADA)4, (KADA)4, or (RAEA)4.

[0027] The present invention also provides the application of the above-mentioned amphiphilic peptide hydrogel biostabilizer in the preparation of bone regeneration drugs.

[0028] Meanwhile, the present invention also provides the application of the above-mentioned amphiphilic peptide hydrogel biostabilizer in the preparation of bone regeneration-promoting drugs in a high-sugar microenvironment.

[0029] Furthermore, the amphiphilic peptide hydrogel biostabilizer simultaneously binds Ca²⁺ + It adsorbs AGEs and integrates them with ECM to promote bone regeneration in a high-sugar microenvironment.

[0030] Compared with the prior art, the present invention has the following beneficial technical effects:

[0031] 1. This invention establishes a quantitative evaluation system for hydrogel mesoscopic mechanical steady state, using DTR=CV. output / CV input Quantifying the degree of dispersion transfer from pathological perturbation input to mesoscopic mechanical output transforms "biostabilization" from a qualitative description into a calculable, comparable, and cross-material-verifiable engineering indicator.

[0032] 2. This invention discloses a biaffinity peptide hydrogel biostabilizer, ViscoClamp. ViscoClamp is not a traditional passive scaffold, but rather a mesoscopic biostabilizer. On the one hand, it buffers chronic glycation load through AGEs affinity modules; on the other hand, it inhibits viscoelastic runaway drift through network clamping, while simultaneously preserving cell-mediated matrix remodeling, collagen fiber reorganization, and Ca²⁺. + The relevant mineralization deposition capacity avoids the drawbacks of overly rigid materials that may hinder cell migration and bone matrix maturation. ViscoClamp can interpenetrate and integrate with MSC-deposited ECM, maintaining stable viscoelasticity in the pericellular microdomain, compatible with matrix remodeling and mineralization, coupling pathological glycation load with adaptive buffering, and its biological stability meets the aforementioned dispersion transfer ratio.

[0033] 3. This invention relates to the application of a biaffinity peptide hydrogel biostabilizer in the preparation of bone regeneration drugs, addressing the clinical challenges of high-glucose jawbone regeneration by proposing a treatment approach shifting from "enhancing osteogenic stimulation" to "maintaining osteogenic niche homeostasis." Existing research on high-glucose bone regeneration often emphasizes anti-inflammatory, antioxidant, pro-angiogenic, or osteogenic factor supplementation. However, if the pericellular biomechanical baseline continues to drift, osteogenic signals may still fail due to a lack of stable integration background. Taking high-glucose jawbone defects as a scenario, this invention incorporates AGEs chemical disturbances and masticatory functional load into the same pathological framework, effectively addressing the core issues of unstable efficacy, long healing periods, and insufficient bone quality in clinical treatment.

[0034] 4. The application of the biaffinity peptide hydrogel biostabilizer of this invention in the preparation of bone regeneration drugs constructs a closed-loop mechanism from material input to tissue regeneration output, encompassing aspects such as local AGEs capture, ViscoClamp / AGEs / Col I co-localization, collagen fiber structure restoration, AFM mesoscopic viscoelastic stabilization, Integrin / Fak mechanotransduction reconstruction, Runx2 osteogenic transcriptional activation, and immune-vascular ecological improvement. This cross-scale mechanism research helps address the issue of unstable osteogenic treatment efficacy in high-glycemic environments, achieving efficient and robust repair of diabetes-related jawbone defects under chronic glycation and functional mechanical loads, and providing a mechanistic basis for subsequent combined growth factor, cell therapy, or scaffold implantation.

[0035] 5. This invention relates to the application of the amphiphilic peptide hydrogel biostabilizer in the preparation of bone regeneration drugs, proposing a micro-mechanical homeostasis reset (MMHR) material paradigm. This paradigm shifts the therapeutic target from traditional material bulk parameter optimization to the maintenance of mesoscopic viscoelastic homeostasis around cells, which is actually integrated into the cell. The core therapeutic target is the 1-50 μm viscoelastic microenvironment around cells, which is actually perceived by the cells. It emphasizes the importance of maintaining mesoscopic mechanical homeostasis for determining the osteogenic fate of MSCs in chronic disease states. This paradigm does not replace biochemical osteoproliferation but rather serves as a "mechanical background therapy" to prolong the effective time window of osteogenic stimulation, reduce response dispersion, and improve the robustness of bone regeneration in high-glucose microenvironments. It is expected to bridge the scale gap between the macroscopic properties of materials and the microscale mechanical perception of cells, providing a new and widely applicable paradigm for tissue regeneration in chronic microenvironments such as diabetes, inflammation, fibrosis, and aging. Attached Figure Description

[0036] Figure 1This is a schematic diagram illustrating the synthesis and characterization of ViscoClamp in an embodiment of the present invention; wherein, A is a representative analytical HPLC chromatogram and LC-MS spectrum of synthesized ViscoClamp, B is a micro-Fourier transform infrared (micro-FTIR) characterization of ViscoClamp, and C is a representative macroscopic morphology, contact angle measurement, and hydrogel microstructure diagram characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of 1% and 6% (w / v) ViscoClamp hydrogels.

[0037] Figure 2 This is a schematic diagram illustrating the amphiphilicity of ViscoClamp in an embodiment of the present invention; wherein, A represents the quantitative determination of ViscoClamp and Ca² using isothermal titration calorimetry (ITC). + A) is the binding affinity map of ViscoClamp and AGEs, B) is the binding affinity map of ViscoClamp and AGEs quantified by fluorescence polarization (FP) experiment, C) is the TEM image of ViscoClamp after incubation with increasing concentrations of AGEs, and D) is the colocalization map of FITC-labeled ViscoClamp (6%) and Cy3-labeled AGEs (0.1 mg / mL) after binding, as shown by confocal laser scanning microscopy (CLSM).

[0038] Figure 3 This is a schematic diagram illustrating the cell compatibility of ViscoClamp in an embodiment of the present invention; wherein, A is a representative immunofluorescence image of Ki67 and Tunnel staining after MSCs have been incubated with ViscoClamp for 24 h, and B is Ki67. + and Tunel + Quantitative cell mapping.

[0039] Figure 4 This is a schematic diagram of the mechanical characterization and parameter extraction of ViscoClamp in an embodiment of the present invention; wherein, A is a schematic diagram of mechanical characterization and parameter extraction based on atomic force microscopy (AFM), and B is a schematic diagram of the biological stabilization concept.

[0040] Figure 5 The diagram shows the viscoelastic output of ViscoClamp in this embodiment of the invention; wherein, A is the AFM-derived viscoelastic parameter diagram of a single gel unit after incubation with increasing concentrations of AGEs, B is the frequency scan rheological diagram of 6% ViscoClamp after incubation with a specified concentration of AGEs, and C is the level of soluble AGEs in the culture supernatant of MSCs.

[0041] Figure 6 This is a graph showing the stabilized output index of ViscoClamp in an embodiment of the present invention.

[0042] Figure 7 This is a schematic diagram illustrating the time robustness of ViscoClamp in an embodiment of the present invention; wherein, A is a schematic diagram of the biostability assessment in the 0–72 h time domain, B is the AFM-derived viscoelastic parameters of a single gel unit measured at a specified time point, and C is the time-resolved CV analysis of the input and output and the corresponding CV. output / CV input ratio.

[0043] Figure 8 This is a diagram of the cell ECM niche failure model in an embodiment of the present invention.

[0044] Figure 9 This is a schematic diagram of ViscoClamp restoring collagen assembly in an embodiment of the present invention; wherein, A is the fluorescence staining of collagen I (Col I) in the MSC-derived matrix and the quantification of Col I fibrillary length, diameter and Col I positive area; B is the confocal imaging of FITC-labeled ViscoClamp (green) and Cy3-labeled AGEs (red) on the MSC surface, and phalloidin / DAPI counterstaining and Imaris three-dimensional reconstruction are performed.

[0045] Figure 10 This is a transcriptome sequencing analysis diagram from an embodiment of the present invention.

[0046] Figure 11 This is a ViscoClamp-supported cell mineralization diagram in an embodiment of the present invention; wherein, A is a cryo-electron microscopy image showing Ca²⁺ at 0, 6, 12, and 24 h after incubation of ViscoClamp with CaCl₂. + Deposition details, B represents Ca²⁺ in the cryo-electron microscopy image. + Quantification of positive area fraction: C represents the three-dimensional topology of AFM tapping mode after incubation of ViscoClamp and CaCl2 for 24 h under conditions with or without AGEs; D represents the Ca²⁺ obtained from the AFM morphology. + Combined with intensity heatmap analysis.

[0047] Figure 12 This is a diagram of ViscoClamp rescuing MSCs for osteogenic differentiation in an embodiment of the present invention; wherein, A is the Alizarin Red S (ARS) staining and Col I immunofluorescence of MSCs on days 7, 14 and 21 of osteogenic differentiation, and B is the quantitative analysis of the absorbance of the ARS extract at 570 nm and the quantitative analysis of the ARS-Col I colocalization intensity.

[0048] Figure 13This is a schematic diagram of a rat mandibular bone defect model injected with AGEs in an embodiment of the present invention.

[0049] Figure 14 This is an example of ViscoClamp improving the repair of mandibular defects in rats in this invention. A shows a representative micro-CT image of a 2-month-old rat mandibular defect; B shows micro-CT quantitative BMD, BV / TV, BS / BV, Tb.N, Tb.Th, and Tb.Sp; C shows sequential fluorescent labeling (calcein, alizarin red, and tetracycline) and quantitative new bone formation at weeks 2–4 and 4–8; D shows representative Masson trichrome staining, Van Gieson (VG), and H&E staining of the mandibular defect area at 1 month; E shows histological morphological quantification of new bone formation (new bone area / defect area); F and G show Von Kossa staining showing the quantitative fraction of mineral and calcium deposition within the defect; H and I show immunofluorescence staining of AGEs in the mandibular defect (H), and quantitative AGEs fluorescence intensity and AGEs positive area fraction (AGEs positive area / defect area) (I); J and K show CLSM. Immunofluorescence co-staining of CD73 and Alp in mandibular bone defects (J). Quantitative analysis of the correlation between CD73 and Alp signals based on ImageJ, and CD73... + Alp + Double-positive cell abundance (K), LO is the immunofluorescence staining of osteocalcin (Ocn) and Col I in a 1-month mandibular bone defect (L, N), and the intensity of Ocn and Col I is quantified (M, O).

[0050] Figure 15 This is a transcriptome pathway analysis diagram of ViscoClamp treatment in an embodiment of the present invention; wherein, A is a transcriptome pathway network analysis comparing ViscoClamp and Ctrl in AGEs-injected rats, B is a GSEA diagram of the collagen / ECM gene set, C is a heatmap of selected differentially expressed genes, and D is a GSEA diagram of the bone development, bone mineralization, and mesenchymal cell differentiation pathways.

[0051] Figure 16This is a diagram showing the safety assessment of ViscoClamp treatment in rats injected with AGEs in this embodiment of the invention. A represents representative H&E staining of major organs in AGEs-injected rats two months after treatment in the saline (Ctrl) and ViscoClamp groups; B represents blood biochemistry and complete blood count results two months after treatment; C represents H&E staining of major organs in healthy rats after intragastric administration of 10 times the dose of ViscoClamp for 7 consecutive days, with saline as a control; and D represents blood biochemistry and complete blood count results after 7 consecutive days of intragastric administration.

[0052] Figure 17 This is a diagram of a New Zealand white rabbit model injected with AGEs in an embodiment of the present invention.

[0053] Figure 18 This is a schematic diagram illustrating the effect of ViscoClamp promoting new bone regeneration in the mandible of rabbits injected with AGEs in an embodiment of the present invention; wherein, A is a representative micro-CT image of a 1-month-old rabbit mandibular defect and quantitative data of BMD, BV / TV, BS / BV, Tb.N, Tb.Th and Tb.Sp, and B is a representative Masson trichrome staining, VG and H&E staining of the 1-month-old mandibular defect area, and quantitative data of new bone formation.

[0054] Figure 19 This is a schematic diagram illustrating the improvement of mandibular bone defect repair in rabbits treated with ViscoClamp in an embodiment of the present invention; wherein, A and B are co-staining of CD73 and Alp immunofluorescence in the mandibular bone defect on CLSM imaging (A), based on ImageJ quantitative correlation of CD73 and Alp signals, and CD73 + Alp + Double-positive cell abundance (B), C and D are AGEs immunofluorescence staining in mandibular bone defects (C), and AGEs fluorescence intensity and AGEs positive area fraction (AGEs positive area / defect area) are quantified (D), E and H are Ocn and Col I immunofluorescence staining in mandibular bone defects 1 month after treatment (E and G), and Ocn and Col I intensity are quantified (F and H).

[0055] Figure 20The images shown are from an embodiment of the present invention and illustrate the improvement of skull defect repair in rabbits using ViscoClamp. A represents the micro-CT analysis and quantification of skull defects 3 months after AGEs injection in rabbits (Ctrl vs. ViscoClamp). B represents the histological staining of skull defects 3 months after treatment (Masson trichrome staining, VG, and H&E). C represents the morphological quantification of new bone formation tissue. D represents the immunofluorescence staining of AGEs in skull defects 3 months after treatment, with quantification of AGEs fluorescence intensity and AGEs-positive area fraction.

[0056] Figure 21 This is a diagram showing the safety of ViscoClamp treatment in a New Zealand white rabbit model injected with AGEs in an embodiment of the present invention. A represents representative H&E staining of major organs in the AGEs-injected rabbit one month after ViscoClamp treatment; B represents blood biochemistry and routine blood counts one month after treatment; C represents representative H&E staining of major organs three months after ViscoClamp treatment; and D represents blood biochemistry and routine blood counts three months after treatment.

[0057] Figure 22 This is an illustration of ViscoClamp improving bone defect repair in hyperglycemic beagle dogs in an embodiment of the present invention. A is a schematic diagram of the hyperglycemic beagle bone defect model; B is the blood glucose level measured 2 weeks after STZ injection in beagle dogs; C is the serum AGEs level measured by ELISA 2 weeks after STZ injection; D and E are in vivo micro-CT analysis and quantification of extraction socket defects in hyperglycemic beagle dogs 2 months after treatment; F and G are representative Masson trichrome staining, VG, and H&E staining of extraction sockets 2 months after treatment, with quantification of new bone formation (G).

[0058] Figure 23 This is a diagram showing the safety of ViscoClamp treatment in hyperglycemic beagle dogs in an embodiment of the present invention; where A is the H&E staining of major organs in beagle dogs 2 months after treatment, and B is the blood biochemistry and blood routine indicators of beagle dogs 2 months after treatment.

[0059] Figure 24 These are images illustrating the repair of bone defects in hyperglycemic rhesus monkeys using ViscoClamp in an embodiment of the present invention. A shows a schematic diagram of the hyperglycemic rhesus monkey bone defect model, with blood glucose and serum AGEs levels measured 2 weeks after STZ injection. B shows micro-CT analysis and quantification of mandibular bone defects in hyperglycemic rhesus monkeys 1 week after treatment. C shows micro-CT analysis and quantification of mandibular bone defects in hyperglycemic rhesus monkeys 1 month after treatment.

[0060] Figure 25 This is a safety test chart of ViscoClamp treatment in hyperglycemic rhesus monkeys in an embodiment of the present invention; wherein, A is the electrocardiogram and major organ ultrasound of the rhesus monkeys before and 1 month after treatment, and B is the blood biochemistry and blood routine indicators of the rhesus monkeys 1 month after treatment. Detailed Implementation

[0061] I. Design and Synthesis of Bisamiphilic Peptide Hydrogel Biostabilizer (ViscoClamp)

[0062] The design of the amphiphilic peptide hydrogel biostabilizer of this invention mainly includes the following steps: First, the mineralization motif of the casein-rich (Statherin) fragment DSSEEKF is referenced, and phosphorylation-simulated substitution (SS→DD) is introduced to generate DDDEEKF; compared with the natural motif, this sequence increases Ca²⁺. + Affinity proxy index. Subsequently, this invention tandemly linked DDDEEKF with the (RADA)4 block to balance the binding and gelation feasibility of AGEs-HSA (AGE-modified human serum albumin). Then, an alanine-to-cysteine ​​scan was performed on the (RADA)4 block, predicting that replacing the terminal alanine with a cysteine ​​resulted in the strongest mechanical locking effect, thus obtaining the cysteine-stabilized sequence DDDEEKF-RADARADARADARADC. Finally, using solid-phase peptide synthesis based on Fmoc (9-fluorenylmethoxycarbonyl protecting group), a hydrogel containing the cysteine-stabilized sequence was prepared, yielding an amphiphilic peptide hydrogel biostabilizer named ViscoClamp, which exhibits high affinity for Ca²⁺. + It has a dual affinity for AGEs.

[0063] In this embodiment, the (RADA)4 block is used as the AGEs adsorption module, which has a better AGEs adsorption effect. In other embodiments of the present invention, (KADA)4 or (RAEA)4 can also be used as the AGEs adsorption module. Similarly, the terminal amino acid of the AGEs adsorption module is replaced with cysteine ​​by cysteine ​​scanning to make it have a better mechanical locking effect, but its effect is not as good as that of (RADA)4.

[0064] II. Experimental Methods

[0065] 1. Design, preparation and characterization of ViscoClamp peptides

[0066] 1.1 Solid-phase peptide synthesis

[0067] ViscoClamp peptide was synthesized using the standard Fmoc solid-phase peptide synthesis method in a solid-phase reactor on dichlorotriphenylmethyl chloride resin (10 g; loading 1.0 mmol / g). The resin was swollen sequentially in dichloromethane (DCM, 10 mL) and N,N-dimethylformamide (DMF, 10 mL) for 10 min each, followed by filtration. For each coupling cycle, the corresponding Fmoc protected amino acid (33 mmol) and N,N-diisopropylethylamine (DIPEA, 100 mmol, dissolved in DMF) were added, and the reaction was carried out with shaking at 30 °C for 120 min. After coupling, the resin was washed 5 times with DCM and 5 times with DMF. Fmoc deprotection was performed twice with 20% (v / v) piperidine / DMF at 30 °C for 10 min each time, followed by 5 washes with DCM and 5 washes with DMF. Deprotection was confirmed by the ninhydrin test (blue color development).

[0068] 1.2 Liquid Chromatography-Mass Spectrometry (LC-MS)

[0069] Crude ViscoClamp monomeric peptides were purified by preparative reversed-phase HPLC using a Gemini NX-C18 column (Phenomenex; 5 μm, 110 Å, 150 × 30 mm). Mobile phase A consisted of water containing 0.1% (v / v) trifluoroacetic acid (TFA), and mobile phase B consisted of acetonitrile containing 0.1% (v / v) TFA. A linear gradient of 5–60% mobile phase B was applied at 30.0 mL / min over 50 min. The collected fractions were lyophilized, and peptide purity was assessed by LC-MS on a Waters binary LC system using a water / acetonitrile system containing 0.1% (v / v) formic acid at a flow rate of 50 μL / min. Mass spectrometry was performed using electrospray ionization positive ion full scan mode.

[0070] 1.3, Microscopic FTIR

[0071] The secondary structure characteristics of lyophilized ViscoClamp monomeric peptides were assessed by micro Fourier transform infrared spectroscopy (Bruker VERTEX70, Germany).

[0072] 1.4 Gel morphology and plasticity

[0073] ViscoClamp monomeric peptides were directly dissolved in phosphate-buffered saline (PBS) to prepare ViscoClamp hydrogels at final concentrations of 1% and 6% (w / v). The gelation process was recorded using the inverted test tube method. To test the plasticity of ViscoClamp, a 6% (w / v) gel was prepared and used for writing letters.

[0074] 1.5, Contact Angle Measurement

[0075] Static contact angle measurements were performed using a contact angle meter (KRÜSS DSA100, Germany) with a 50 µL droplet of ViscoClamp (1% or 6% (w / v)).

[0076] 1.6 Transmission Electron Microscopy (TEM)

[0077] To observe the nanoscale morphology, advanced glycosylation end products (AGEs; BIOSS, bs-1158P) were added to 6% (w / v) ViscoClamp to achieve final concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, or 1.6 mg / mL. The samples were then dropped onto a carbon-coated copper grid and imaged using TEM (Talos L 120C G2, Thermo Fisher Scientific, USA).

[0078] 1.7 Scanning Electron Microscopy (SEM)

[0079] 1% and 6% (w / v) ViscoClamp gels were lyophilized, mounted on the SEM stage, and sputtered with approximately 8 nm of platinum before imaging, followed by imaging using a SEM (GeminiSEM 500, Zeiss, Germany).

[0080] 1.8, Isothermal titration calorimetry (ITC)

[0081] The binding of ViscoClamp to CaCl2 was measured at 25 °C using an isothermal titration calorimeter (ITC200, Switzerland). Both ViscoClamp (20 µM) and CaCl2 (200 µM) were prepared with ultrapure water. After thoroughly cleaning the sample cell and syringe, a blank titration with water was performed to confirm baseline stability. The ViscoClamp solution was carefully added to the sample cell, avoiding the introduction of air bubbles, and the CaCl2 solution was titrated into the sample cell in 20 consecutive injections (2.0 µL each; injection duration 4 s; interval 150 s). The reference power was set to 5 cal / s, and the stirring speed was set to 60 × g. Water was added to the reference cell to maintain thermal equilibrium. Data were analyzed using the instrument's Origin software to obtain the binding isotherm.

[0082] 1.9, Fluorescence Polarization (FP)

[0083] In fluorescence polarization (FP) experiments, FITC was coupled to the N-terminus of ViscoClamp, followed by dialysis, purification, and lyophilization. Binding to AGEs was measured in black 96-well plates using a microplate reader (SpectraMax iD5, Molecular Devices, USA). Serially diluted AGEs were incubated with FITC-labeled 6% (w / v) ViscoClamp at room temperature (final volume 100 µL per well). After 2 h, the AGEs were measured in λ... ex = 470 nm and λ em FP was recorded under the condition of 530 nm.

[0084] Dissociation constant (K) d The following method combines a model with nonlinear regression to obtain the results:

[0085] (1)

[0086] Where FP represents the measured polarization value, F0 is the polarization value when FITC-ViscoClamp exists alone, and F c [Pep] represents the polarization value at saturation, [Pep] represents the final concentration of ViscoClamp, and [AGE] represents the total concentration of AGEs.

[0087] 1.10, Confocal Laser Scanning Microscope

[0088] ViscoClamp and AGEs were labeled with FITC and Cy3 fluorescence, respectively. 6% (w / v) ViscoClamp gels with or without 0.1 mg / mL AGEs were placed on glass slides and imaged using a confocal microscope (Leica TCS SP8STED 3X, Germany) to assess AGEs-dependent changes in gel structure.

[0089] 1.11, Rheological Measurement

[0090] The viscoelastic behavior of 6% ViscoClamp and AGEs (BIOSS, bs-1158P) was characterized using frequency sweep analysis. Different concentrations of AGEs were tested, including 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mg / mL. Frequency sweeps were performed at 25 °C with a constant strain of 1% and an angular frequency (ω) range of 100–0.1 rad / s. Simultaneously, viscosity properties were measured at 25 °C with shear rates ranging from 0.1–100 1 / s. Storage modulus (G′), loss modulus (G″), and loss factor (G″ / G′) were recorded. Repeated frequency sweeps were used to determine the storage modulus and loss modulus to observe their continuous changes.

[0091] 1.12, Cryo-electron microscopy

[0092] The 1% (w / v) ViscoClamp preparation solution was prepared under three conditions: (i) untreated control, (ii) CaCl2 treatment alone (0.1 mg / mL), and (iii) CaCl2 (0.1 mg / mL) combined with AGEs (0.1 mg / mL). Samples were incubated at 4 °C and collected at 0, 6, 12, and 24 h. Aliquots were spread onto a copper grid to form a thin aqueous film, and then rapidly immersed in liquid ethane for cryo-vitrification. Samples were examined using a Talos F200C instrument (FEI, USA) via cryo-electron microscopy.

[0093] 2. Characterization of the multiscale viscoelastic behavior and ultrastructure of ViscoClamp using atomic force microscopy (AFM).

[0094] The mechanical properties and ultrastructure of lyophilized 6% (w / v) ViscoClamp samples were characterized using atomic force microscopy (AFM) (CSI Nano-Observer, France). All measurements were performed using a tapered probe (ACTA, APPNANO, USA) with a nominal resonant frequency f = 300 kHz, a spring constant k = 37 N / m, and a tapered probe half-open angle φ = 18°.

[0095] First, prepare the materials: add gradient concentrations of adsorbate (AGEs) to multiple hydrogels of equal volume, mix well, and let stand for 1-2 hours to obtain multiple sets of test samples.

[0096] Before measurement, the spring constant k was calibrated using the thermal noise method, and the flexural sensitivity s was determined by pressing into a hard glass substrate. For ultrastructural characterization, all samples were scanned in tapping mode at a scan rate of 1 line / s, with each image having a resolution of 512 lines.

[0097] For static mechanical characterization, the probe is indented at a quasi-static velocity of 200 nm / s until a preset maximum force of 300 nN is reached. The indentation depth δ is calculated using the following formula:

[0098] (2)

[0099] Where z represents the z-position of the cantilever beam, and d represents the cantilever beam deflection. To avoid the base effect, the total indentation depth is controlled to be below 2 µm (<10% of the sample thickness). The applied force is calculated using Hooke's law:

[0100] (3)

[0101] static elastic modulus The Sneddon model was used for calculations, which is applicable to the indentation of a tapered probe into soft materials.

[0102] (4)

[0103] Where F represents the applied force, δ represents the indentation depth, and φ is the half-open angle of the conical probe. Therefore, the apparent elastic modulus of the sample can be calculated using the following formula:

[0104] (5)

[0105] Among them, E exp Let ν be the apparent elastic modulus and ν be Poisson's ratio; for incompressible hydrogels, assume ν = 0.5.

[0106] For dynamic viscoelastic characterization, a dynamic creep indentation method was used with a high AFM probe indentation speed (approximately 20 μm / s) to simultaneously capture elastic and viscous properties. After reaching a preset applied force (F = 300 nN), the load was held for 10 s to observe indentation creep. The creep compliance J(t) was defined as the ratio of strain ε(t) to stress σ(t) and calculated based on the Lee-Radok formula.

[0107] (6)

[0108] (7)

[0109] Where H(t) is the Heaviside step function, F(t) represents the load applied to the gel, and F0 represents the preset force. Let t be the indentation depth as a function of time. Equation (6) extends the Sneddon model to viscoelastic materials, allowing the stiffness as a function of time to be inferred from the indentation data.

[0110] To characterize the multiscale viscoelastic behavior of the gel, both a self-similar hierarchical model and a power-law model were used to analyze all dynamic creep results (i.e., the creep compliance J(t) mentioned above). First, we established a two-level self-similar hierarchical model to describe the dynamic creep response of the sample at t>0.1s:

[0111] (8)

[0112] (9)

[0113] The two can be written together:

[0114] (10)

[0115] Among them, based on the gel microstructure, E1 characterizes the stiffness of local gel units, E2 characterizes the stiffness of fibers within the gel, and τ characterizes the gel relaxation time.

[0116] We used this model to fit the creep compliance-time curves in the range of 0.01–0.1 s to obtain the parameters E1, E2, and τ. Subsequently, we calculated the effective viscosity η of the local gel unit of the sample using η=E1×τ.

[0117] Furthermore, to understand the rheological behavior of the samples, a power-law model was used to characterize the creep response of the samples within the same time range (0.1–10 s):

[0118] (11)

[0119] Here, α is the power-law exponent, a relevant index characterizing the time-dependent creep deformation of the sample. When α=0, it indicates that the material is an ideal elastic solid; when α=1, it indicates that the material is an ideal viscous fluid. The slope, i.e., the power-law exponent α, can be obtained by taking the logarithm of the creep compliance-time curve and performing linear fitting.

[0120] 3. Cell experiments

[0121] 3.1 Culture of mesenchymal stem cells (MSCs)

[0122] The mandibles of male rats were aseptically processed to obtain primary mandibular bone marrow mesenchymal stem cells (JBMSCs). The extracted mandibles were briefly sterilized and then stored on ice in PBS containing 3% (w / v) fetal bovine serum (FBS) and antibiotics. After removing attached soft tissue (and teeth if necessary), the bone marrow cavity was accessed by trimming the alveolar region and mandibular ramus. The bone marrow was washed with complete α-MEM (α-MEM containing 10% (w / v) FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin), gently dispersed mechanically by pipetting, filtered through a cell filter, and collected by centrifugation at 800×g for 3–5 min. To improve cell recovery from dense mandible bone, residual bone was further minced into 1–3 mm³ fragments and digested with 0.1% (w / v) type II collagenase at 37°C with shaking for 60 min. After washing, the bone fragments were co-seeded to promote stromal cell migration. Cells were seeded in 100 mm culture dishes and cultured in a humidified 5% (v / v) CO2 incubator at 37°C. After 48–72 h, the cells were washed with PBS and the culture medium was changed to remove non-adherent cells; the medium was then changed every 2–3 days. Cells were passaged at 80–90% confluence (usually 1:2), and the 2nd–3rd passages were used for subsequent in vitro experiments.

[0123] 3.2 SEM Analysis

[0124] MSCs were seeded onto coverslips in 24-well plates and cultured until fully confluent. Cells were exposed to AGEs (0.1 mg / mL) and treated for 24 h with or without 1% (w / v) ViscoClamp. Samples were fixed, dehydrated, dried, sputter-coated with approximately 8 nm of platinum, and then imaged using SEM (GeminiSEM 500, Zeiss, Germany).

[0125] 3.3 Alp Activity Detection

[0126] According to the manufacturer's instructions, the activity of Alp in cell culture medium was quantified using a commercial Alp activity assay kit (NJJCBIO, A059-2-2, China). Alp activity was calculated as follows: Alp activity (U·L⁻¹) -1 = (AT − A0) / (AS − A0) ×100×Cs×7.14, where AT represents the absorbance of the sample, A0 represents the absorbance of the blank well, AS represents the absorbance of the standard solution at 520 nm, and Cs represents the concentration of the standard solution.

[0127] 3.4 Immunofluorescence staining

[0128] MSCs were seeded into confocal culture dishes (1 × 10⁶ per dish). 4 Cells were divided into three conditions: control, AGEs, or AGEs plus 1% ViscoClamp. When the cells reached approximately 80% confluence, the medium was changed to osteogenic induction medium containing 100 nmol / L dexamethasone, 50 mg / mL ascorbic acid, and 1 mmol / L β-glycerophosphate. On days 7, 14, and 21, cells were fixed with 4% (w / v) paraformaldehyde at room temperature for 10 min, permeabilized with 0.3% (w / v) Triton X-100 for 30 min, and blocked with goat serum. Samples were incubated overnight at 4 °C with primary antibodies against collagen I (Col1; Proteintech, 66761), AGEs (MCE, HY-P81087), and Runx2 (CST, 12556S), followed by incubation at room temperature with fluorophore-conjugated secondary antibody (YeasenBio, China) for 1 h. Mineral deposits were visualized using Alizarin Red S (Beyotime, C0148S) as needed. Cell nuclei were counterstained with DAPI for 10 min. Images were acquired using a confocal microscope (Olympus FV3000, Japan), and fluorescence intensity was quantified using ImageJ.

[0129] In co-localization studies, MSCs (1 × 10⁶ per dish) were used. 4 Cells were incubated with FITC-labeled ViscoClamp and Cy3-labeled AGEs for 6 h. Cells were then fixed and permeabilized as described above, blocked with goat serum, and incubated overnight at 4°C with anti-Col1 (Proteintech, 66761). Samples were incubated with YSFluor™ 647 secondary antibody (YeasenBio, 34413ES60, China) for 1 h, counterstained with DAPI for 10 min, and imaged on a confocal microscope (LeicaTCS SP8 STED 3X, Germany).

[0130] 3.5 Western blotting

[0131] MSCs were seeded in 6-well plates (1 × 10⁶ cells per well). 5Cells were lysed (number of cells) and processed as directed. Cells were lysed with ice-cold RIPA lysis buffer containing PMSF for 30 min and centrifuged at 13,800 g for 15 min at 4 °C to remove insoluble debris. Cytoplasmic and nuclear fractions were prepared using the Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent, SM-005-4, USA) according to the manufacturer's instructions. Lysis buffer was denatured at 100 °C for 10 min in SDS loading buffer containing 50 mM DTT, and protein concentration was determined by the BCA method (Beyotime, P0010S, China). Equal volumes of protein were separated by 10% (w / v) SDS-PAGE and transferred to a 0.45 µm PVDF membrane (Millipore, IPFL00010). The membrane was blocked with 5% (w / v) bovine serum albumin at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies Runx2 (CST, 12556S), ALP (HUABIO, ET1601), Lamin A / C (Beyotime, AG2517), and β-actin (PTMBIO, PTM-5018). After washing with TBST, HRP-conjugated secondary antibody (YeasenBio, China) was added and incubated at room temperature for 1 h, followed by development with enhanced chemiluminescence (ECL; Millipore).

[0132] 3.6 RNA Sequencing and Analysis of MSCs

[0133] MSCs were cultured in 100 mm dishes and divided into two groups based on AGE exposure. Total RNA was extracted using TRIzol after 24 h of treatment. RNA-seq libraries were prepared using the NEBNext® Ultra RNA Library Prep Kit for Illumina® (NEB, E7530L, USA) and paired-end sequencing (2 × 150 bp) was performed on the Illumina NovaSeq 6000 platform. Heatmaps and gene set enrichment analyses were generated using Qlucore Omics Explorer 3.2, and pathway analysis was performed using Ingenuity Pathway Analysis (IPA).

[0134] 3.7, Alizarin Red S staining and quantification

[0135] MSCs were prepared at a ratio of 1 × 10⁻⁶ per well. 5Cells were seeded at a density in 24-well plates and divided into multiple groups as needed. When cells reached approximately 80% confluence, the medium was switched to osteogenic induction medium (100 nmol / L dexamethasone, 50 mg / mL ascorbic acid, and 1 mmol / L β-glycerophosphate). Mineralization was assessed on days 7, 14, and 21 using Alizarin Red S staining (Beyotime, C0148S). Mineralized nodules were dissolved in 2% (w / v) cetylpyridinium chloride solution, and absorbance was measured at 570 nm using a SpectraMax 190 (Molecular Devices, USA).

[0136] 3.8 Cytotoxicity Evaluation

[0137] In the imaging-based cytotoxicity assay, MSCs (1 × 10⁶ per well) were used. 4 Cells were treated with 1% (w / v) ViscoClamp for 24 h, fixed, permeabilized with 0.3% (w / v) Triton X-100 for 30 min, and blocked with goat serum. Samples were stained with Ki-67 (CST, 9129S) and labeled using the TUNEL apoptosis detection kit (Beyotime, C1089, China), followed by FITC-conjugated secondary antibody (YeasenBio, China) and nuclear counterstaining with DAPI (1 min). Confocal images were acquired on an Olympus FV3000.

[0138] 4. Animal experiments

[0139] 4.1 Experimental Model

[0140] AGEs Injection Model: AGEs (BIOSS, bs-1158P, China) were administered intraperitoneally as an experimental intervention. AGEs were diluted with sterile PBS to the required working concentration for each experiment. Rats received 20 mg / kg, and New Zealand white rabbits received 10 mg / kg, injected three times a week. At the end of week 4, peripheral blood was collected 24 hours after the last injection, and serum AGEs were quantified using an AGEs detection kit (Abcam, ab238539, USA) according to the manufacturer's instructions. Bone defect surgery was performed the following day. Postoperatively, AGEs were continued to be injected at the same frequency (three times a week) until the study endpoint, at which point samples were collected for subsequent analysis.

[0141] STZ-induced hyperglycemia: Streptozotocin (STZ; Solarbio, S8050, China) was freshly dissolved in 0.05 M sodium citrate buffer (pH 4.5). All animals were fasted for 8 hours prior to STZ administration, but had free access to water. Under anesthesia, beagles were given a single intravenous dose of 40 mg / kg STZ, and rhesus monkeys were given a single intravenous dose of 45 mg / kg STZ. Fasting blood glucose was monitored regularly, and subsequent procedures were initiated after stable hyperglycemia was established.

[0142] Rat mandibular defect model: Four weeks after AGEs injection, rats were anesthetized with inhaled isoflurane. A circular defect (4 mm in diameter; 0.8 mm in depth) was established in the periapical region of the first and second molars. The defect location was first marked with a 4 × 4 mm trephine; when the depth approached approximately 0.8 mm, bone was gently removed with a ball bur until the target defect geometry was achieved. Rinsing with physiological saline was maintained throughout the drilling process. An animal self-control design was used: the left side was given ordinary physiological saline (control), and the right side was given 6% (w / v) ViscoClamp. Two months later, the rats were sacrificed with sodium pentobarbital. Blood was collected for biochemical analysis and serum was separated. The mandible and major organs were harvested for subsequent analysis.

[0143] Rabbit mandibular defect model: Four weeks after AGEs injection, rabbits were anesthetized with isoflurane. After shaving the hair on the sides of the mandible, a skin incision was made below the molars to expose the mandible. A circular defect was created using an 8 mm hollow trephine after thorough rinsing with Hank balanced salt solution, and the base of the defect was smoothed with a 2 mm ball bur, forming a defect with a diameter of 8 mm and a depth of 2 mm. A contralateral design was used, with the left side receiving physiological saline and the right side receiving 6% (w / v) ViscoClamp. One month later, the rabbits were euthanized, and blood samples were collected for biochemical analysis and serum separation. The intact mandible and organs were harvested for further analysis.

[0144] Rabbit skull defect model: Four weeks after AGEs injection, rabbits were anesthetized with isoflurane, and the hair on their skulls was shaved. A midline incision of approximately 50 mm was made along the sagittal suture to expose the periosteum. Within one quadrant of the skull, an 8 mm trephine was used to reduce heat production and establish a circular defect under continuous saline irrigation; subsequently, the base of the defect was smoothed with a 2 mm ball bur, forming a defect with a diameter of 8 mm and a depth of 2 mm. The left defect was treated with saline, and the right defect with 6% (w / v) ViscoClamp. Three months later, the rabbits were euthanized, blood was collected for biochemical analysis, and skull tissues and organs were harvested.

[0145] Beagle tooth extraction model: After establishing hyperglycemia with STZ injection, beagles were anesthetized with sodium pentobarbital, and bilateral mandibular lateral incisors were extracted. Excess blood was removed, and the left extraction socket was treated with normal saline, while the right extraction socket was treated with 6% (w / v) ViscoClamp. Samples were collected two months post-treatment. Blood was collected for biochemical analysis, and serum was separated. Mandibular bone tissue and internal organs were collected for further evaluation.

[0146] Rhesus monkey mandibular defect model: Hyperglycemia was induced in rhesus monkeys by STZ administration. Animals were fasted for 24 h and deprived of water for 12 h preoperatively. Anesthesia was induced with intravenous propofol and maintained with inhaled isoflurane. A mandibular defect (10 × 5 × 4 mm) was created in the posterior molar region. A contralateral design was used: normal saline was administered on the left side, and 6% (w / v) ViscoClamp was administered on the right side, and the gel was slowly and evenly implanted until the defect was completely filled. The gingival wound was closed with non-absorbable sutures, taking care to prevent gel leakage. Postoperatively, ceftiofur sodium (3–5 mg / kg) was administered intramuscularly daily for 7 consecutive days to prevent infection, and the wound was irrigated daily with normal saline containing metronidazole for 7 consecutive days.

[0147] 4.2 Blood Glucose Measurement

[0148] Blood glucose was measured using a glucose oxidase-based blood glucose meter (OneTouch UltraEasy, Johnson & Johnson, USA) according to the manufacturer's instructions. Results are expressed in mmol / L.

[0149] 4.3 Quantitative Analysis of AGEs

[0150] The concentration of AGEs in blood samples and conditioned medium was measured using an ELISA-based assay kit (Abcam, ab238539, USA). Briefly, 50 µL of standard or sample was added to wells coated with AGEs conjugate and incubated for 10 min, followed by 50 µL of diluted anti-AGEs antibody and incubation for 1 h. After washing the plate with 250 µL of 1× wash buffer, 100 µL of diluted HRP-conjugated secondary antibody was added and incubated for 1 h, followed by another wash. 100 µL of TMB substrate solution was added and reacted for 2–20 min. The reaction was terminated with 100 µL of stop solution, and the absorbance was immediately read at 450 nm. The sample concentration was calculated using a standard curve based on the blank-corrected absorbance values.

[0151] 4.4 Micro-computed tomography (micro-CT)

[0152] Ex vivo samples from mice, rats, rabbits, and dogs were scanned on a high-resolution micro-CT system (Xradia 610 Versa, Carl Zeiss, Germany) with parameters of 100 kV, 110 µA, and an exposure time of 500 ms. Rhesus monkeys underwent in vivo CT imaging at 1 week and 1 month post-surgery using an AX3000-D scanner (Always Imaging, China) with tube voltages of 160 or 180 kV, tube currents of 0.1–0.5 mA, and integration times of 1 s. Three-dimensional reconstructed images were generated, and regions of interest (ROIs) containing bone defects / extraction sockets were defined on the reconstructed models. Standardized bone morphological parameters, including bone mineral density (BMD), bone volume fraction (BV / TV), bone surface area fraction (BS / BV), trabecular bone number (Tb.N), trabecular bone spacing (Tb.Sp), and trabecular bone thickness (Tb.Th), were quantified using the manufacturer's software.

[0153] 4.5, Histological staining

[0154] Decalcified bone sections were stained with hematoxylin-eosin (H&E; Baso, BA4097 / BA4098, China), Masson trichrome staining (Solarbio, G1340, China), or Van Gieson staining (Baso, BA4084A, China) according to the kit protocol. Sections were digitized using an automated slide scanner (Pannoramic DESK, 3D HISTECH, Hungary).

[0155] 4.6, Immunofluorescence of bone sections

[0156] Sections were subjected to heat-induced antigen retrieval and blocked with normal goat serum at room temperature for 1 h. Samples were incubated overnight at 4 °C with primary antibodies against CD73 (Invitrogen, PA5-85958), Alp (HUABIO, ET1601), osteocalcin (Ocn; Proteintech, 23418), AGEs (Sigma, MABN1837), and collagen I (Col1; Proteintech, 66761). After incubation with fluorophore-conjugated secondary antibody (YeasenBio, China) for 1 h, cell nuclei were counterstained with DAPI for 10 min, and images were acquired using an automated scanning system (AxioScan Z, Zeiss, Germany).

[0157] 4.7 Sequential fluorescent labeling

[0158] To assess mineral deposition and regeneration dynamics, rats were sequentially injected with calcein green (CA, 20 mg / kg), alizarin red (AR, 30 mg / kg), and tetracycline (TE, 25 mg / kg) at weeks 2, 4, and 8. Three days after tetracycline injection, animals (n = 3) were sacrificed, and maxillary bone specimens were fixed in 4% (w / v) formaldehyde for 48 h, embedded in resin, sectioned (approximately 150 µm), and ground to approximately 40 µm. Fluorescent labeling was observed using a confocal microscope (Leica SP8 DIVE, Germany) at excitation / emission settings of 488 / 517 nm (calcein), 543 / 617 nm (alizarin red), and 405 / 580 nm (tetracycline).

[0159] 4.8 Von Kossa staining of hard tissue sections

[0160] Undecalcified mandibular hard tissue sections were stained using the Von Kossa kit (Solarbio, G3282, China). Sections were exposed to silver nitrate solution under strong light for 15–60 min, rinsed with distilled water for 1 min, and then treated with sodium thiosulfate for 2 min to terminate the reaction. Imaging was performed using an automated slide scanner (Pannoramic DESK, 3D HISTECH, Hungary).

[0161] 4.9 RNA Sequencing and Analysis

[0162] Mandibular bone tissue was collected on day 7 after treatment with 6% (w / v) ViscoClamp, and total RNA was extracted using TRIzol reagent (Invitrogen, 15596026CN, USA). Libraries were prepared using the NEBNext® Ultra RNA Library PrepKit for Illumina® (NEB, E7530L, USA), and paired-end sequencing (2 × 150 bp) was performed on the Illumina NovaSeq 6000 platform. Expression heatmaps and gene set enrichment analyses were generated in Qlucore Omics Explorer 3.2, followed by genome enrichment and gene ontology analyses.

[0163] 4.10, Toxicology

[0164] For acute oral toxicity, healthy male SD rats were administered ViscoClamp by gavage daily for 7 consecutive days at a dose 10 times the experimental dose, followed by sacrifice. Organs were collected and fixed in 4% (w / v) paraformaldehyde. Whole blood was collected for complete blood cell count (CBC) and serum chemistry tests, and tissues were routinely processed and stained with H&E.

[0165] For treated rats, rabbits, and beagle dogs, animals were sacrificed after treatment, and CBC markers (RBC, WBC, LYMPH, NEUT, PLT, and HGB) and serum biochemical markers (ALT, AST, BUN, and creatinine) were assessed. Major organs were fixed in 4% paraformaldehyde and subjected to H&E staining after routine treatment.

[0166] For macaques, CBC and serum chemistry were assessed before treatment and one month after ViscoClamp implantation; electrocardiograms and ultrasound were also monitored to record the condition of the heart, liver, spleen and kidneys.

[0167] 4.11 Statistical Analysis

[0168] Statistical analyses were performed using SPSS 22.0 and GraphPad Prism 10. Normality was assessed using the Shapiro-Wilk test. Comparisons between two independent groups were performed using either a two-tailed unpaired Student t-test or a paired Student t-test. Data are presented as mean ± SD, with SD shown in error bars. Significance thresholds were set as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Each experiment was independently repeated at least three times. Unless otherwise stated, sample sizes were not predetermined through power calculations, no data points were excluded, and experiments were not randomized or blinded.

[0169] III. Experimental Results

[0170] 1. ViscoClamp Feature Detection

[0171] The ViscoClamp prepared above was validated by mass spectrometry. Figure 1 (A). FTIR spectra show characteristic amide and thiol absorption bands, supporting the successful introduction of cysteine. Figure 1 B); after RP-HPLC purification, the purity reached 98% ( Figure 1 (B). ViscoClamp can form a gel at ≥1% (w / v). Figure 1 The 6% (w / v) hydrogel exhibited good integrity and injectability, and also possessed favorable rheological behavior. Figure 1 (C).

[0172] ITC verified its compatibility with Ca² + The structural basis of the combination (K) d =3.15 ±1.13 μM) Figure 2 Fluorescence polarization results showed that it had a strong binding ability with AGEs-HSA, with an apparent dissociation constant of 1018 ± 224 nM. Figure 2 (B). Consistent with adsorption-induced network recombination, TEM showed that ViscoClamp nanofibers gradually aggregated and underwent structural rearrangement after the addition of AGEs. Figure 2 Confocal imaging of Cy3-AGEs mixed with FITC-ViscoClamp showed that the two co-aggregated within the gel matrix. Figure 2 D).

[0173] Finally, cell compatibility experiments showed that 1% (w / v) ViscoClamp did not affect MSC proliferation or induce apoptosis. Figure 3 This supports its use as a cell-contact biomaterial.

[0174] 2. ViscoClamp compresses discrete saccharification inputs into stable viscoelastic outputs with a lower discreteness transfer ratio.

[0175] To examine whether ViscoClamp functions as a biological stabilizer, this invention formulates stabilization as an input-output problem: the gradient concentration of AGEs is used as the perturbation input, and the cell-scale viscoelastic parameters obtained by atomic force microscopy (AFM) are used as the output. Figure 4 (A and B). This invention combines the quasi-static indentation method to extract the apparent elastic modulus (E). exp The Sneddon model was used, and the creep compliance (J(t); Lee-Radok formula) curve was obtained by dynamic creep indentation. Then, a hierarchical model and power-law fitting were used to decompose it to obtain multi-scale viscoelastic parameters corresponding to different AGEs concentrations, including a set of compact cell-scale mechanical readings such as gel relaxation time (τ), effective viscosity (η), power-law exponent (α), local gel unit stiffness (E1), and fiber network stiffness (E2). Figure 4 (A). In other embodiments of the present invention, other static elasticity testing methods may also be used to extract the apparent elastic modulus.

[0176] To quantitatively evaluate mesoscopic mechanical steady state, this invention uses the coefficient of variation (CV = standard deviation (SD) / mean) as a dimensionless index of dispersion. CV is defined as follows: input The coefficient of variation for AGEs concentration is given, and the output parameter m belongs to {E}. exp , α, τ, E1, η,E2},CVoutput This represents the overall dispersion.

[0177] The concentration sequence A = {ai} corresponding to the gradient concentrations of AGEs, where ai represents the concentration of AGEs, and CV input = SD(A) / mean(A).

[0178] The coefficient of variation (CV) of the output parameters is obtained by calculating the standard deviation / mean of each parameter in output parameter m using SD(m) / mean(m). m Coefficient of variation (CV) m Including E exp Given the coefficients of variation of α, τ, E1, η, and E2, calculate E. exp The arithmetic mean of the coefficients of variation of α, τ, E1, η, and E2 is used to obtain the overall dispersion CV. output .

[0179] Mesoscopic mechanical stability is evaluated by the dispersion transfer ratio (DTR), where DTR = CV. output / CV input ×100%, CV output Much lower than CV input This means that large input fluctuations are compressed into a narrow output range. In this invention, if 4.5% ≤ DTR ≤ 5%, the hydrogel is considered to have mesoscopic mechanical stability; otherwise, the mesoscopic mechanical stability of the hydrogel is insufficient. The determination of the DTR range in this invention follows these principles:

[0180] (1) Physiological threshold constraint: Under normal physiological conditions, the inherent dispersion of viscoelastic parameters around cells is 8%-12%. The CV fluctuation is input to AGEs. input Under pathological conditions of ≥200%, the output dispersion coefficient (CV) can only be guaranteed when DTR ≤ 5.0%. output ≤200%×5.0%=10%, completely returning to physiological levels.

[0181] (2) Functional failure boundary: When DTR>5.0%, the output viscoelastic dispersion exceeds 10%, which will lead to significant heterogeneity in cell differentiation direction, disordered ECM assembly, and ultimately loss of the core function of stabilizing the microenvironment.

[0182] (3) Biodynamic requirements: The core of hydrogels to achieve mesoscopic stability is "stable but not rigid", rather than completely eliminating mechanical fluctuations. When DTR < 4.5%, the viscoelasticity of the material is too uniform and fixed, which will hinder the dynamic remodeling process of cell-deposited ECM and inhibit cell migration, proliferation and mechanotransduction.

[0183] (4) Material manufacturability constraints: DTR below 4.5% requires peptide purity ≥ 99.5% and crosslinking deviation < 2%, which will increase the synthesis cost by more than 3 times and significantly increase the batch-to-batch difference, making it unfeasible for clinical translation.

[0184] This embodiment uses a wide range of AGEs concentrations (0-25.6 mg / mL) to test ViscoClamp, and AFM measurements are performed after lyophilization of the gel. With increasing AGEs load, η and τ show a slight decreasing trend, while E... exp α, E1, and E2 remain essentially unchanged. Figure 4 B and Figure 6 A). The bulk rheology of the gel after exposure to AGEs also did not show substantial changes in elastic behavior or force transmission characteristics. Figure 5 Consistent with its preferential regulation of viscosity (stress relaxation) behavior, ViscoClamp supplementation reduced the level of soluble AGEs in MSC culture supernatant, decreasing detectable AGEs by ≥50% within 4 h. Figure 5 (C).

[0185] CV input from AGEs input The dispersion exceeds 200%, while the dispersion of the output parameters is significantly lower (CV). output E exp =9.1%, α=9.7%, τ=15.3%, E1=3.7%, η=14.3%, E2=4.3%. Combining these outputs, we obtain the discrete transfer ratio (CV). output / CV input =4.7%, corresponding to a decrease of approximately 20 times in the dispersion from saccharification input to viscoelastic output ( Figure 6 ).

[0186] To verify time robustness, mesoscopic mechanical steady state was analyzed at multiple time points after AGEs exposure. This invention performed mechanical spectrum analysis at 6, 12, 24, 48, and 72 hours after AGEs exposure. Figure 7 (A). The time series output reproduced the single-time-point pattern ( Figure 7 (B), and the time-dependent discreteness transfer ratio remains essentially unchanged (CV) output / CV input =4.75% Figure 7 (C). In summary, these data indicate that CV can be... output / CV input Established as a quantitative characteristic of mesoscopic mechanical steady state, the ViscoClamp of this invention can maintain stable viscoelastic output under highly varying saccharification loads, achieving biostabilization through a low dispersion transfer ratio.

[0187] 3. ViscoClamp salvages osteogenic differentiation while still allowing mineralization by restoring collagen assembly and pericellular mechanics.

[0188] Having demonstrated that ViscoClamp can compress discrete glycosylation inputs into a narrowly distributed viscoelastic output, this invention further investigates how mechanical stabilization translates into a biologically executable effect. Since MSC fate decisions are filtered through the surrounding ECM microdomain, biological stabilizers not only need to resist bulk mechanical drift but must also interpenetrate and hybridize with the matrix on which MSCs are deposited, thereby stabilizing the cell-perceived mechanical microenvironment. Figure 8 Therefore, this invention establishes a glycosylation-driven niche failure model at the ECM level. MSCs are first cultured to confluence to promote sufficient ECM deposition, followed by administration of AGEs (0.1 mg / mL), with the option of adding 1% (w / v) ViscoClamp.

[0189] Collagen staining further revealed that AGEs caused significant collagen disorder, resulting in shortened fiber length, reduced fiber number, and increased apparent fiber diameter; while the addition of ViscoClamp significantly improved collagen arrangement and polymerization characteristics. Figure 9 To determine whether this rescue originates from physical integration at the cell surface, this invention uses FITC-labeled ViscoClamp and Cy3-labeled AGEs (A). Figure 9 (B). Three-dimensional confocal reconstruction showed that ViscoClamp and AGEs were strongly colocalized on the MSC surface, with a relative coefficient R of 0.95 and an overlap area of ​​over 81%.

[0190] To link structural rescue with molecular programming, this invention performed transcriptome analysis on MSCs under AGEs glycation load with and without ViscoClamp. Transcriptome sequencing combined with gene set enrichment analysis (GSEA) showed that, compared with AGEs alone, ViscoClamp-treated MSCs were enriched in collagen formation / organization and ECM remodeling pathways. Figure 10 Overall, these results indicate that ViscoClamp can adsorb AGEs and integrate them into the pericellular matrix, restoring collagen structure and mechanics at the cellular scale, thus establishing a mechanistic bridge between stable viscoelastic output and stable cellular niche input.

[0191] Since craniofacial regeneration requires not only early osteogenic initiation but also continuous matrix maturation, this invention next evaluates whether ViscoClamp, while stable and hybridized with the ECM, remains compatible with mineral deposition and osteogenic progression under glycosylation conditions. This invention first challenges ViscoClamp with CaCl2 under conditions with and without AGEs, and tracks mineral deposition over time. Cryo-electron microscopy shows Ca²⁺… +It accumulates gradually on ViscoClamp over time; it is worth noting that, compared to individual Ca² + In comparison, AGEs further enhanced mineral deposition ( Figure 11 (A and B). AFM topological imaging confirmed that surface mineral accumulation increased after ViscoClamp treatment ( Figure 11 (C and D).

[0192] Subsequently, this invention investigated whether ViscoClamp could rescue osteogenic differentiation of MSCs under glycosylation conditions. During osteogenic induction, Alizarin Red S (ARS) staining and collagen staining at days 7, 14, and 21 showed that AGEs significantly inhibited mineralized nodule formation and reduced ARS-collagen I (Col I) co-localization; co-treatment with 1% ViscoClamp largely restored mineral deposition and matrix tissue (…). Figure 12 (A and B). In summary, these data indicate that ViscoClamp can alleviate glycation stress while effectively restoring the microenvironment for mineralization and osteogenic activation.

[0193] 4. Surrounding micromechanical stabilization remodels early regeneration processes and improves mandibular bone defect repair in glycation-challenged rats.

[0194] Next, this invention investigates whether pericellular viscoelastic clamping under sustained glycation load can be transformed into robust in vivo regeneration. This invention uses an AGEs-injected rat mandibular defect model to evaluate ViscoClamp. Bilateral mandibular defects were established 3 days after the last injection, and patients were immediately treated with either saline (Ctrl) or ViscoClamp. Figure 13 ).

[0195] At 2 months, micro-CT analysis showed significantly enhanced bone regeneration within the defect treated with ViscoClamp. Figure 14 A). Quantitative assessment showed that, compared with the saline control, bone mineral density (BMD) increased by 1.2 times, bone volume fraction (BV / TV) increased by 1.3 times, trabecular bone number (Tb.N) increased by 1.9 times, and trabecular bone thickness (Tb.Th) increased by 1.5 times, while BS / BV decreased by about half and Tb.Sp decreased by 69%. Figure 14 (B). Sequential fluorescent labeling showed an accelerated mineral deposition rate: compared to the control, the mineralization rate of the ViscoClamp group increased by 1.5 times in weeks 2–4 and by 87% in weeks 4–8. Figure 14 C). Histological staining (Masson, VG, H&E) further confirmed a significant increase in bone filling and maturation, with the area of ​​newly formed bone increasing approximately twofold. Figure 14D and E). Von Kossa staining also showed a 1.7-fold increase in mineral deposition in the ViscoClamp Formation (D and E). Figure 14 (F and G). Immunofluorescence analysis provided cell- and matrix-related evidence for this repair phenotype. ViscoClamp enables CD73... + Alp + The abundance of double-positive osteogenic progenitor cells increased 3.7-fold. Figure 14 H and I). Meanwhile, the AGEs signal in the defect area increased (area 4.7 times; intensity 6.6 times) Figure 14 The levels of osteogenic matrix markers such as J and K were elevated, accompanied by increased levels of Ocn (area 16.9 times; intensity 3.5 times) and Col I (area 6.3 times; intensity 3.9 times). Figure 14 (LO). This pattern is consistent with the local isolation of AGEs in the gel-ECM hybrid niche, rather than systemic clearance.

[0196] To link structural regeneration with tissue-level transcriptional programs, transcriptome pathway analysis and GSEA of 1-week-old tissues showed enrichment of collagen formation / fibrous tissue and ECM remodeling pathways, as well as bone development, mineralization, and mesenchymal differentiation programs, in ViscoClamp-treated defects. Figure 15 ).

[0197] Hematological, serum biochemical, and organ histological results at 2 months showed no significant toxicity, supporting its systemic biosafety. Figure 16 (A and B). To further assess the safety margin of oral exposure, healthy Sprague Dawley rats were administered 10 times the dose of ViscoClamp by gavage for 7 consecutive days; hematology, serology, and H&E histology remained within the normal range ( Figure 16 (C and D).

[0198] 5. ViscoClamp improves the repair of mandibular and cranial defects in rabbits while maintaining long-term biocompatibility.

[0199] To validate efficacy in a larger craniofacial context, this invention established a New Zealand white rabbit model of AGEs injection. After establishing bilateral mandibular defects, saline (Ctrl) and ViscoClamp were administered to the contralateral sides (left Ctrl; right ViscoClamp; n=6). Figure 17 ).

[0200] At 1 month, micro-CT showed significant enhancement of mandibular bone regeneration in the ViscoClamp group. Figure 18Specifically, BMD increased by 38%, BV / TV increased by 2.1 times, Tb.N increased by 1.2 times, and Tb.Th increased by 1.1 times, while BS / BV decreased by 35% and Tb.Sp decreased by 58%. Histological analysis (Masson, VG, and H&E) confirmed increased bone filling and maturation, and quantitative analysis showed a 1.9-fold increase in new bone area. Figure 18 B).

[0201] Immunofluorescence ( Figure 19 Further display of CD73 + Alp + Double-positive osteogenic progenitor cells increased (3.3-fold), local AGEs signaling was enhanced (3.1-fold area; 2.5-fold intensity), and osteogenic matrix markers, including Ocn (7.3-fold area; 1.5-fold intensity) and ColI (4.4-fold area; 2.6-fold intensity), were elevated. These results further support the mechanism that ViscoClamp locally captures glycation load while stabilizing the osteogenic permissive niche.

[0202] In the same AGEs-injected rabbit model, this invention further investigated a skull defect model to evaluate bone formation under different craniofacial geometries and healing environments. At 3 months, micro-CT analysis showed that ViscoClamp significantly enhanced skull regeneration. Figure 20 Specifically, BMD increased by 57%, BV / TV increased by 1.1 times, Tb.N increased by 1.2 times, and Tb.Th increased by 28%, while BS / BV decreased by 21% and Tb.Sp decreased by 59%. Histological examination (Masson, H&E, and VG staining) confirmed improved defect bridging and matrix deposition, with a 1.1-fold increase in new bone area. Figure 20 (B and C). ViscoClamp group AGEs stained more strongly (area 5.5 times; intensity 7.2 times). Figure 20 The D), consistent with the continuous local isolation effect during long-term healing.

[0203] Hematological / biochemical and organ histological studies confirmed biocompatibility, with no adverse changes observed at 1 month. Figure 21 (A and B). At 3 months, normal organ histology and hematology / biochemistry results further support its long-term biosafety. Figure 21 (C and D).

[0204] 6. Large animal and non-human primate models demonstrate its robustness under hyperglycemia and functional load.

[0205] Small animal models can establish mechanistic rationale; however, craniofacial reconstruction requires validation under near-human-scale defect geometry, slower remodeling dynamics, and sustained masticatory loads, factors that are even more challenging under hyperglycemic conditions. Therefore, this invention evaluates ViscoClamp in STZ-induced hyperglycemic large animals and non-human primates, models where the mechanical environment and regeneration timescale more closely approximate clinical craniofacial repair.

[0206] In 12-month-old male beagle dogs, a single intravenous injection of STZ (40 mg / kg) successfully induced hyperglycemia, such as... Figure 22 As shown in A. Compared with the solvent control, fasting blood glucose levels increased 1.6-fold, and serum AGEs levels increased 3.3-fold. Figure 22 (B and C). A paired design was used to establish mandibular incisor extraction socket models: the left extraction socket was treated with physiological saline (Ctrl), and the right extraction socket was immediately filled with 6% ViscoClamp (n=3 per group). Figure 22 (A). At 2 months, in vivo micro-CT analysis showed significantly enhanced bone regeneration in the extraction socket of the ViscoClamp group. Figure 22 D and E). BMD (36%), BV / TV (42%), Tb.N (43%), and Tb.Th (30%) were significantly increased, while BS / BV (42%) and Tb.Sp (46%) were significantly decreased. Histological examination (Masson, VG, and H&E) confirmed increased trabecular formation and maturation, with a 1.5-fold increase in new bone area. Figure 22 (F and G).

[0207] No detectable toxicity was observed in systemic hematology / biochemistry and organ histology results. Figure 23 (A and B) support the biosafety of this treatment in the oral environment of large animals.

[0208] Subsequently, the invention turned to the rhesus monkey model; among all preclinical systems, this model most closely resembles the human craniofacial anatomy and loading patterns. Figure 24 (A). In 5-year-old male rhesus monkeys, a single intravenous administration of STZ (45 mg / kg) induced hyperglycemia, resulting in a nearly 90% increase in fasting blood glucose and a 2.9-fold increase in serum AGEs. A mandibular bone defect (10×5×4 mm) was created near the molar region using a paired design: the left defect was treated with normal saline (Ctrl), and the right defect with 6% ViscoClamp. Micro-CT scans one week post-surgery showed accelerated early bone formation in the defect treated with ViscoClamp (A). Figure 24Compared with the paired control, BMD increased by 29%, BV / TV increased by 5.6 times, Tb.N increased by 2.4 times, and Tb.Th increased by 64%, while BS / BV decreased by 47% and Tb.Sp decreased by 65%. At 1 month, CT scans still showed that ViscoClamp treatment of the defect had more adequate bone filling and maturation. Figure 24 (C). Given that posterior mandibular defects with a depth ≥4 mm inevitably carry the risk of damaging adjacent tooth roots, the molars in the defect area were lost during the follow-up period and were not visible on a 1-month CT scan.

[0209] Systemic monitoring (including electrocardiogram, organ ultrasound, and hematology / biochemistry) showed no signs of cardiotoxicity or organ abnormalities. Figure 25 Overall, these data suggest that ViscoClamp can maintain functional performance and biocompatibility under conditions of high glycemia, chronic glycation load, and craniofacial mechanical load—a key prerequisite for driving the translation of MMHR biostabilizers into clinically relevant craniofacial repair.

[0210] By establishing a preclinical validation chain from small animals to non-human primates, the efficacy of the materials was not only validated in rat and rabbit jaw / skull defects, but also further developed into extraction socket models in hyperglycemic beagle dogs and mandibular defect models in rhesus monkeys, covering common clinical scenarios in oral medicine such as extraction socket healing, jaw defect repair, and dynamic mastication load. This multi-species, multi-scale, and multi-timepoint evidence of efficacy and safety will significantly enhance the credibility of translating this project into clinical applications for jaw regeneration in hyperglycemic patients.

[0211] This invention redefines mechanical regulation as a materials problem through MMHR: it requires maintaining the mechanics of actual cellular integration—i.e., mesoscopic cell-to-pericellular viscoelasticity—rather than optimizing static bulk stiffness. This invention treats pericellular viscoelastic homeostasis as a controllable design variable. MMHR complements biochemical osteogenic induction strategies and provides a physical basis for explaining why baseline drift in mechanotransduction under chronic stress makes osteogenic stimuli temporally vulnerable. This invention chooses glycation as a rigorous proof-of-concept perturbation because AGEs accumulate persistently, have significant mechanical consequences, and are difficult to remove; furthermore, dynamic loading in the craniomaxillofacial region further destabilizes the niche. Based on these results, cross-species bone repair and mineralization improvement demonstrate that stabilizing pericellular mechanics can improve the persistence and reproducibility of osteogenic regeneration trajectories under chronic microenvironment drift.

[0212] This invention proposes a novel theoretical framework for MMHR (Mechanomodulation of Bone Regeneration), redefining the mechanical regulation of bone regeneration as a mesoscopic material design problem. It focuses on pericellular viscoelastic homeostasis rather than traditional bulk stiffness optimization, elucidating the physical mechanisms underlying the time-dependent fragility of osteogenic stimuli under chronic stress. The design approach for dual-affinity peptide hydrogel biostabilizers is particularly suitable for MMHR materials because these materials must co-encode resusceptibility, disease input buffering, and drift inhibition, while also integrating with a continuously remodeling living ECM. More broadly, modularity enables adaptability: by replacing sensing / tuning modules, MMHR materials can be reconfigured to buffer other chronic perturbations, such as inflammatory mediators, oxidative stress, or fibrosis-related crosslinking, without altering the output target of pericellular mechanical homeostasis. While this invention has been validated in craniofacial regeneration challenging with glycation, MMHR is intended as a generalizable therapeutic axis for disease states where chronic microenvironment drift disrupts fate program stability. Mesoscopic stabilizers such as ViscoClamp can be deployed as mechanical “background” therapies to prolong the effective duration of biochemical cues and reduce response variability, and can be used in combination with growth factor delivery, cell therapy, or implantable scaffolds. By demonstrating that pericellular viscoelastic homeostasis can be maintained without impairing the maturation of the mineralized matrix, this study establishes mesoscopic mechanical homeostasis as an actionable target and provides a blueprint for designing biostabilizers that function in vivo and in disease-modified ECMs.

Claims

1. A method for evaluating the mesoscopic mechanical homeostasis of a hydrogel, characterized in that, Includes the following steps: Step 1: Add gradient concentrations of adsorbate to multiple hydrogels of equal volume, mix thoroughly, and let stand for x hours to obtain multiple sets of test samples; x takes the value of 1 to 2. Step 2: Measure the apparent elastic modulus E of the sample to be tested. exp The power-law exponent α, gel relaxation time τ, local gel unit stiffness E1, effective viscosity η, and fiber network stiffness E2; Step 3, Define CV input The coefficient of variation of the adsorbate concentration is calculated from the standard deviation / mean of the adsorbate concentration described in step 1. Define that the output parameter m belongs to {E} exp Given the set of adsorbates at gradient concentrations { , α, τ, E1, η, E2}, calculate the standard deviation / mean of the output parameter m, and obtain the coefficient of variation (CV) of the output parameter m. m Coefficient of variation (CV) m Including E exp The coefficients of variation of α, τ, E1, η, E2; Calculate E exp The arithmetic mean of the coefficients of variation of α, τ, E1, η, and E2 is used to obtain the overall dispersion CV. output ; Step 4, based on CV output / CV input ×100% yields the Dispersion Transfer Ratio (DTR); Step 5: If 4.5%≤DTR≤5%, the hydrogel is considered to have mesoscopic mechanical stability; otherwise, the hydrogel has insufficient mesoscopic mechanical stability.

2. The method for evaluating the mesoscopic mechanical steady state of a hydrogel according to claim 1, characterized in that: It also includes step 6, in which steps 2-5 are repeated for multiple groups of test samples at different time points after x hours of placement in step 1, to obtain the corresponding mesoscopic mechanical stability results, in order to test time robustness.

3. The method for evaluating the mesoscopic mechanical steady state of a hydrogel according to claim 1 or 2, characterized in that, Step 2 is as follows: Multiple groups of samples were examined using atomic force microscopy, and the creep compliance was obtained by combining it with dynamic creep indentation. Simultaneously, the apparent elastic modulus E was obtained using quasi-static indentation. exp ; All creep compliances were analyzed using a self-similar hierarchical model and a power-law model. Creep compliance-time curves within the range of 0.01–0.1s were fitted to obtain the local gel unit stiffness E1, fiber network stiffness E2, and gel relaxation time τ. The effective viscosity η is calculated based on the local gel unit stiffness E1 and the gel relaxation time τ. The power-law exponent α can be obtained by taking a single logarithm of the creep compliance-time curve and performing linear fitting.

4. A biaffinity peptide hydrogel biostabilizer, wherein the biostabilizer has mesoscopic mechanical stability as evaluated by any one of claims 1-3, characterized in that: Includes amino acid sequences; The amino acid sequence includes a calcium-binding module and an AGEs adsorption module connected in sequence, and the terminal alanine A of the AGEs adsorption module is replaced with cysteine ​​C.

5. The amphiphilic peptide hydrogel biostabilizer according to claim 4, characterized in that: The calcium binding module is DDDEEKF; The AGEs adsorption module is (RADA)4, (KADA)4, or (RAEA)4.

6. The use of the amphiphilic peptide hydrogel biostabilizer of claim 4 in the preparation of a bone regeneration drug.

7. The use of the amphiphilic peptide hydrogel biostabilizer of claim 4 in the preparation of a bone regeneration-promoting drug in a high-glucose microenvironment.

8. The application of the amphiphilic peptide hydrogel biostabilizer according to claim 7 in the preparation of a bone regeneration-promoting drug in a high-glucose microenvironment, characterized in that: The amphiphilic peptide hydrogel biostabilizer simultaneously binds Ca²⁺. + It adsorbs AGEs and integrates them with ECM to promote bone regeneration in a high-sugar microenvironment.

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