A magnetically responsive growth plate repair scaffold, and methods of making, using, and assessing growth potential thereof

Abstract

The application provides a magnetically responsive growth plate repair bracket and a preparation method, application and growth potential evaluation method thereof, and belongs to the technical field of biological medical materials. The magnetically responsive growth plate repair bracket constructed by the application can respond to an external static magnetic field, and provide a cell in the bracket with a direction and size controllable biomimetic mechanical load, namely axial micro-mechanical stimulation, so as to regulate the cell fate. Compared with a traditional growth plate replacement bracket, the magnetically responsive growth plate repair bracket has the following two advantages: first, the composition is simple and the biocompatibility is good, which can provide the cell with axial micro-mechanical stimulation, support cell adhesion, migration and chondrogenic differentiation; and second, the static magnetic field is used to apply magnetic force stimulation to the magnetically responsive growth plate repair bracket, and the unique advantages of minimally invasive, remote and in-situ application are used, so that the in-vivo application limitation of a traditional mechanical loading mode is broken, and a new direction is provided for growth plate injury repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a magnetically responsive growth plate repair scaffold, its preparation method, application, and growth potential assessment method. Background Technology

[0002] The growth plate (epithelial plate) in children is a cartilaginous tissue located between the metaphysis and epiphysis of long bones, responsible for the longitudinal growth of the skeleton. Its structure is highly ordered, consisting of resting areas, proliferative areas, hypertrophic areas, and calcified areas. Chondrocytes are arranged in a columnar pattern, proliferate actively, and are highly sensitive to mechanical signals. During skeletal development, the growth plate continuously promotes bone length increase through endochondral ossification, making it an indispensable growth and development tissue.

[0003] However, children's bones are not yet fully developed, and trauma, infection, and iatrogenic procedures (such as tumor resection and fracture fixation) can easily damage the growth plate structure. After injury, local chondrocytes become disordered, and blood supply becomes abnormal, leading to the abnormal formation of bone bridges—that is, bone tissue crossing the growth plate to connect the metaphysis and epiphysis. The presence of bone bridges can restrict or even completely block the normal expansion of the growth plate, causing an imbalance in growth rates on both sides, ultimately leading to permanent sequelae such as limb shortening and angular deformities (such as genu varum and genu valgum). These complications not only affect the child's motor function but also impose a heavy medical and economic burden on the family.

[0004] Currently, the mainstream clinical treatment for growth plate injury is bone bridge resection combined with filling with inert materials (such as fat, silicone, polymethyl methacrylate, etc.) to physically isolate bone bridge regeneration. However, this approach has limited efficacy, a high long-term recurrence rate, and cannot restore the original longitudinal growth capacity of the growth plate. The reason for this is that existing treatment strategies neglect the crucial role of the biomechanical microenvironment in maintaining the ordered structure of the growth plate and regulating chondrocyte phenotype. Bone growth is inseparable from mechanical stimulation: appropriate dynamic loads can maintain the columnar arrangement of chondrocytes and promote the differentiation of mesenchymal stem cells into cartilage; conversely, the absence or abnormality of biomechanical signals leads to disordered growth plate tissue structure, accelerated chondrocyte apoptosis, and abnormal matrix mineralization. Therefore, there is an urgent need to develop a filling scaffold that can provide biomimetic biomechanical stimulation to the damaged area to reconstruct a biomechanical microenvironment conducive to growth plate regeneration and restore its developmental potential.

[0005] In recent years, studies have found that secondary ossification centers exhibit a mechanical buffering effect during growth, placing the growth plate cartilage in a micromechanical environment along the bone growth direction, i.e., axial micromechanics (eLife 9 (2020) e55212). However, traditional mechanical loading methods (such as piezoelectric ceramics and hydraulic actuation) are mainly limited to in vitro experiments. In clinical practice, they suffer from drawbacks such as difficulty in in vivo application, significant trauma, and difficulty in long-term maintenance, which greatly limits their practical application in in situ repair of growth plates.

[0006] Therefore, there is an urgent need to develop a filling scaffold that can provide biomimetic mechanical stimulation to the damaged area in order to reconstruct a mechanical microenvironment conducive to growth plate regeneration and restore its developmental potential. Summary of the Invention

[0007] The purpose of this invention is to provide a magnetically responsive growth plate repair scaffold, its preparation method, application, and growth potential assessment method. The prepared magnetically responsive growth plate repair scaffold has good biocompatibility, supports cell adhesion, migration, and chondrogenic differentiation. At the same time, after being combined with a static magnetic field, the biological effects are further enhanced by stimulating the axial micromechanical properties of cells, breaking through the limitations of traditional mechanical loading methods in vivo application and providing a new direction for growth plate damage repair.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a magnetically responsive growth plate repair scaffold, the raw materials for which the magnetically responsive growth plate repair scaffold is prepared include 80-120 parts of dry GelMA, 4-6 parts of MNPs, and 0.05-0.2 parts of TGF-β3.

[0009] Preferably, the raw materials for preparing the magnetic response growth plate repair scaffold include 100 parts of dry GelMA, 5 parts of MNPs, and 0.1 parts of TGF-β3.

[0010] The present invention also provides a method for preparing the magnetically responsive growth plate repair scaffold as described in claim 1, comprising the following steps: (1) Dissolve the dry GelMA and heat and stir to obtain a GelMA hydrogel solution; (2) Dissolve MNPs in the GelMA hydrogel solution of step (1) to obtain a magnetically responsive GelMA solution; (3) TGF-β3 was added to the magnetically responsive GelMA solution to obtain the precursor of the magnetically responsive growth plate repair scaffold; (4) Take the precursor of the magnetic response growth plate repair scaffold, heat it to liquid state, and photocrosslink it to obtain the magnetic response growth plate repair scaffold.

[0011] Preferably, in step (1), the temperature of the heating and stirring is 35~45℃.

[0012] Preferably, in step (4), the heating temperature is 35~38°C.

[0013] Preferably, in step (4), the conditions for photocrosslinking are: irradiation with 405 nm light for 20~40 s.

[0014] The present invention also provides an application of the above-described magnetic response growth plate repair scaffold or the magnetic response growth plate repair scaffold prepared by the above-described preparation method in growth plate repair and preparation of growth plate repair scaffolds.

[0015] This invention also provides a method for evaluating the growth potential of a magnetically responsive growth plate repair scaffold, comprising the following steps: (1) The degree of limb shortening and angular deformity is detected by calculating the amount of limb shortening and the angle of angular deformity on the operated side. The formula used is as follows: The amount of limb shortening on the operated side:

[0016] In the formula: δL — Tibial shortening amount / mm; L 1—Length of the healthy side tibia / mm; L 2—Tibia length on the operated side / mm; Angle of angular deformity:

[0017] In the formula: θ —Abnormal angle / °; α 1—Healthy side MPTA / °; α 2—Operative side MPTA / °; (2) The recovery effect of growth potential in the defect area can be intuitively evaluated by the change of magnetic-epistem distance.

[0018] The beneficial effects of this invention compared to the prior art are as follows: (1) Based on a deep understanding of the mechanical sensitivity characteristics of growth plate chondrocytes and bone marrow mesenchymal stem cells, this invention constructs a magnetically responsive growth plate repair scaffold. This magnetically responsive scaffold can respond to an external static magnetic field and provide cells within the scaffold with biomimetic mechanical loads that are directional and size controllable, i.e., axial micromechanical stimulation, thereby regulating cell fate. Compared with traditional growth plate replacement scaffolds, the magnetically responsive scaffold of this invention has the following two advantages: First, it has a simple composition and good biocompatibility, and can provide axial micromechanical stimulation to cells, supporting cell adhesion, migration, and chondrogenic differentiation; second, it uses a static magnetic field to apply magnetic stimulation to the magnetically responsive growth plate repair scaffold, and with its unique advantages such as minimally invasive, remote, and in situ application, it breaks through the limitations of traditional mechanical loading methods in vivo application, providing a new direction for growth plate damage repair.

[0019] (2) The magnetically responsive scaffold of this invention, in conjunction with a static magnetic field, can not only induce cartilage regeneration in situ, significantly alleviating limb shortening and angular deformities, but also greatly enhance the chondrogenic differentiation efficiency of BMSCs in vitro. Experiments have shown that, under the synergy of a static magnetic field, the magnetically responsive growth plate repair scaffold of this application can induce cartilage regeneration in situ, significantly alleviating limb shortening and angular deformities of the affected tibia in rabbits. In addition, the static magnetic field can significantly enhance the chondrogenic differentiation efficiency of BMSCs in vitro. Compared with traditional growth plate scaffolds, this invention rapidly recruits stem cells and induces cartilage regeneration in the early stages of injury, overturning the industry's understanding that severe growth plate damage of supercritical size is irreparable.

[0020] (3) This invention, through the synergistic effect of the magnetic response scaffold and the static magnetic field, can protect the normal growth plate tissue in the vicinity of the injury and avoid growth arrest caused by trauma. The scaffold maintains the homeostasis of the local microenvironment by creating a suitable mechanical environment and biochemical signals, which provides the necessary conditions for cartilage regeneration and long-term reconstruction of the growth plate structure. It is a key mechanism for the damaged growth plate to restore its growth function.

[0021] (4) This invention is the first to propose using the "magnetic-epithelial distance" (MGPD, i.e., the distance from the implanted magnetic patch to the epiphyseal plate) as a visualized and quantitative indicator of growth potential recovery after growth plate injury. This method can be implemented in routine X-ray examinations without the need for additional complex equipment, and can dynamically monitor the growth rate and functional status of the damaged growth plate. This not only represents a breakthrough from simple "structural repair" to "functional reconstruction" evaluation, but also provides an intuitive, simple, and highly valuable detection method for real-time clinical assessment of the growth and development of children with epiphyseal injuries. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This document describes the preparation and morphology of T@GM and T@MGM in Example 1 of the present invention, where A is a schematic diagram of the preparation of the T@MGM magnetic response growth plate repair scaffold; and B is the general morphology of T@GM and T@MGM. Figure 2 Different concentrations of GelMA in Example 1 of this invention GFP+ hBMSCs morphology; Figure 3 In the hydrogels of different concentrations of MNPs in Example 1 of this invention GFP+The proliferation and apoptosis of hBMSCs are shown in Figure A, where TUNEL and Ki67 immunofluorescence staining are used; B shows the apoptosis rate of cells in the three hydrogel scaffolds; and C shows the percentage of proliferating cells in the three hydrogel scaffolds. Figure 4 The storage modulus and loss modulus of the magnetically responsive hydrogel scaffold before crosslinking at different temperatures in Experiment Example 1 of this invention; Figure 5 This is an example of the injectability characterization of the magnetic response growth plate repair scaffold before crosslinking in Experiment Example 1 of the present invention; Figure 6 This is a diagram showing the effect of T@MGM being attracted by a magnet in Experiment Example 1 of this invention; Figure 7 The microstructure and elemental distribution of the magnetic response growth plate repair scaffold in Experimental Example 1 of this invention are shown below. A represents the microstructure of T@GM and T@MGM; B represents the porosity statistics of T@GM and T@MGM; C represents the elemental distribution of T@MGM; and D represents the X-ray energy dispersive spectroscopy (EDS). Note: T@MGM is the example and T@GM is the comparative example. Figure 8 This refers to the measurement of the magnetic induction intensity of the magnet used in Experiment Example 2 of the present invention; Figure 9 This is a biocompatibility test of the magnetically responsive growth plate repair scaffold in Experiment Example 2 of the present invention, where A represents the morphology of GFP+hBMSCs after co-culturing with each group of hydrogel scaffolds for 2 and 9 days; and B represents the distance at which GFP+hBMSCs grow into the hydrogel scaffold. Figure 10 This is a statistical analysis of the SOX9, COL2A1 and ACAN immunofluorescence staining and the corresponding positive cell percentages of each group of hydrogel scaffolds after 16 days of chondrogenic induction culture in Experiment Example 2 of the present invention. Figure 11 The images shown are of the normal growth plate and intraoperative photographs of each group in Experiment Example 3 of this invention, where A is the normal growth plate; B is the intraoperative photograph of the Blank group; C is the intraoperative photograph of the T@GM group; D is the intraoperative photograph of the T@MGM group; and E is the intraoperative photograph of the T@MGM+S group. Figure 12 The images shown are of the initial tibial length, MPTA analysis, and transverse and coronal views of the rabbits on the day of surgery in Experiment Example 3 of this invention. A represents the statistical difference in tibial length between the two sides; B represents the statistical difference in MPTA between the two sides; C represents the transverse view of the modeled tibia; and D represents the coronal view of the modeled tibia. Figure 13 This is a SEM observation of the injury area 2 weeks after modeling a proximal growth plate defect in the rabbit tibia in Experiment Example 3 of this invention. Figure 14 HE and Safranin O-Fixed Green staining were performed 2 weeks after the rabbit proximal tibial growth plate defect model was created in Experiment Example 3 of this invention. Figure 15This is an X-ray evaluation of the repair effect of proximal tibial growth plate defect in rabbits 8 weeks after modeling. A shows X-ray images of the tibia in each group; B shows a schematic diagram of tibial length and MPTA measurement; C shows a statistical chart of shortening deformity; and D shows a statistical chart of angular deformity. Figure 16 The micro-CT evaluation of bone microstructural parameters in the growth plate damage area of ​​rabbits after 8 weeks of proximal tibial growth plate defect modeling in Experiment Example 3 of this invention is presented as a statistical analysis. In this analysis, A represents the micro-CT images of each group of samples; B represents the bone volume (BV) of the growth plate damage area in each group; C represents the bone density (Tb.N) of the growth plate damage area in each group; and D represents the bone density (BMD) of the growth plate damage area in each group. Figure 17 This is a statistical analysis of the growth plate height in the adjacent area of ​​the injury in each group of samples 8 weeks post-operation in Experiment Example 3 of this invention; Figure 18 The statistical results of the appearance and deformity of the tibia sample 8 weeks after surgery in Experiment Example 3 of this invention are shown, where A is the tibial X-ray at different time points; B is the statistical analysis of MGPD at different time points. Figure 19 HE staining of rabbit heart, liver, and kidney sections from each group in Experiment Example 3 of this invention. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Example 1 Embodiment 1 of the present invention provides a method for preparing a magnetically responsive growth plate repair scaffold, the specific steps of which are as follows: Take 0.6 g of 10% GelMA dry matter (purchased from EFL), add 6 mL of PBS buffer, heat and stir at 40°C to dissolve into a 10% (w / v) GelMA solution (GM). Continue by adding 30 mg of MNPs (purchased from Maclean's) to prepare a 5 mg / mL magnetically responsive GelMA solution (MGM). Sterilize the solution in a light-protected bottle by heating in a 60°C water bath for 30 min and then storing at -20°C. Before use, add TGF-β3 according to the actual dosage, mix by pipetting, and encapsulate with TGF-β3.

[0030] TGF-β3 was dissolved in GM and MGM solutions to form T@GM and T@MGM solutions, respectively, with a TGF-β3 concentration of 0.1 μg / μl. Uncrosslinked T@MGM was heated to a liquid state in a 37°C water bath and photocrosslinked by irradiation with 405 nm light from a portable curing light source for 20 s, resulting in a stable and bioactive magnetically responsive growth plate repair scaffold, T@MGM.

[0031] Using T@GM solution as a control, the solution was heated to a liquid state in a 37°C water bath and then irradiated with 405 nm light from a portable curing light source for 20 s to complete photocrosslinking, thus obtaining T@GM. The texture of the crosslinked gel was then observed, and the results are as follows: Figure 1 As shown.

[0032] Figure 1 The results show that at 37℃, T@GM and T@MGM are liquid before crosslinking and solid after photocrosslinking. T@GM is translucent, while T@MGM is grayish-black due to the addition of MNPs, with uniform color. The crosslinked magnetic response growth plate repairs the scaffold with uniform texture, no obvious bubbles or cracks, and good structural integrity, maintaining a stable three-dimensional morphology without external force.

[0033] Comparative Example 1 Comparative Example 1 of this invention adopted the method of Example 1 to detect the effect of the ratio of GelMA to MNPs on the proliferation effect. The specific steps are as follows: Products with different proportions were prepared according to the dosages shown in Table 1.

[0034] Table 1. Ratio of GelMA to MNPs

[0035] use GFP+ hBMSCs were co-cultured with each group of hydrogel scaffolds, and the results are as follows: Figure 2 , 3 As shown.

[0036] Figure 2 The results showed that cells screened using 10% GelMA as the substrate were in better condition. Sections were prepared from three groups of samples: 10% GM, 10% 5MGM, and 10% 10MGM. Figure 3 TUNEL and Ki67 staining was performed to observe cell apoptosis and proliferation within the hydrogel scaffold, showing that a MNP concentration of 5 mg / mL was optimal for cell growth. Therefore, the magnetically responsive growth plate repair scaffold substrate was determined to be 10% GelMA with 5 mg / mL MNPs.

[0037] Experimental Example 1 Experimental Example 1 of this invention comprehensively characterized the magnetically responsive growth plate repair scaffold prepared in Example 1. The specific steps are as follows: (1) Rotational rheological test Uncrosslinked T@MGM was placed on an Austrian-Anton Paar-MCR 102e rotational rheometer, and the mode was set to oscillation / dynamic. Oscillatory amplitude scanning was performed at 37°C with an amplitude range of 2–10 Hz and a constant strain of 3% to determine the storage modulus (G') and loss modulus (G''). Next, an oscillatory temperature ramp was performed at a constant frequency of 5 Hz with a strain of 3% and a temperature range of 4–60°C to determine the changes in G' and G''. The results are as follows: Figure 4 As shown.

[0038] The precursor of the magnetically responsive growth plate repair scaffold should be able to be injected in situ in a liquid state at a suitable temperature, thereby fully filling the damaged area of ​​the growth plate. Figure 4 The results showed that the solid-liquid phase transition of the stent precursor occurred at 32.21±0.00577℃. The optimal temperature for stent implantation is 37℃, at which point the stent precursor is in a liquid state, meeting the injection requirements.

[0039] (2) Injectability test Take the uncrosslinked T@MGM solution and heat it to a liquid state in a 37°C water bath. Protect from light, draw it out with a 1 mL syringe and inject it onto a disposable sheet following the planned path. Then irradiate with 405 nm light for 20 s to complete photocrosslinking. The results are as follows. Figure 5 As shown.

[0040] Figure 5 The results showed that the uncured magnetically responsive growth plate repair scaffold could be extruded from a 1 mL syringe at 37°C and arranged along a preset path. After irradiation with 405 nm light for 20 s, it could be cross-linked and cured in situ, exhibiting an "XJTU" pattern.

[0041] (3) Magnetic responsiveness test T@MGM should exhibit magnetic responsiveness, enabling manipulation by an external static magnetic field. A cross-linked T@MGM scaffold sample was placed in the center of a 10 cm diameter cell culture dish, and an appropriate amount of PBS buffer was added to reduce friction between the hydrogel block and the dish bottom. A magnet was placed on the side of the culture dish, and the movement of the scaffold due to magnetic attraction was observed. The results are as follows... Figure 6 As shown.

[0042] Figure 6 The results show that the T@MGM magnetic response growth plate repair scaffold accelerates towards the small magnet after being attracted by a static magnetic field, visually demonstrating its excellent magnetic responsiveness.

[0043] (4) Scanning electron microscopy for microstructure detection and elemental analysis The cross-linked T@MGM was placed in a -80°C freezer overnight, and then freeze-dried for 12 hours the following day before liquid nitrogen brittle fracture. After gold sputtering, the samples were placed in the sample chamber according to the markings and their positioning adjusted. The observation field was determined, and the sample cross-section was observed and photographed at 1000x and 2000x magnification, respectively. The results are as follows: Figure 7 As shown. The scanning electron microscope is a ThermoFisher Scientific ApreoS from the USA.

[0044] GelMA, as a modified product of gelatin, possesses good biocompatibility, a suitable porous structure, and high porosity, providing an ideal environment for cell adhesion and proliferation. To verify whether the magnetically responsive growth plate repair scaffold T@MGM with introduced MNPs still retains its original excellent structural characteristics, scanning electron microscopy (SEM) was used to observe the internal structure of the T@MGM sample and compared it with T@GM without MNPs. Figure 7The results showed that both groups of samples had porous mesh structures in their cross-sections, which facilitates nutrient diffusion and the removal of metabolic waste, and provides spatial support for cell ingrowth and migration. Statistical analysis revealed that the porosity of T@GM was 46.93% ± 7.19%, and that of T@MGM was 45.47% ± 4.20%, with no significant difference between the two groups. T@MGM effectively retained its high porosity. Further elemental distribution mapping of T@MGM showed that iron in the MNPs was uniformly distributed within the hydrogel scaffold. This preliminarily ensured that T@MGM experienced uniform stress within the static magnetic field, while also guaranteeing a homogeneous microenvironment for endogenous cells within the magnetically responsive growth plate repair scaffold.

[0045] Experimental Example 2 Experimental Example 2 of this invention evaluated the recruitment of magnetically responsive growth plate repair scaffolds and the induction of hBMSCs chondrogenic differentiation. The specific steps are as follows: (1) Recovery of hBMSCs hBMSCs were purchased from OriCell. After being removed from the cryovials in liquid nitrogen, they were quickly transferred to a 37°C water bath for thawing. During thawing, the cryovials should be kept upright, with only the bottom in contact with the water bath, to avoid contamination from liquid entering the tube opening. Gently shake the cryovials to accelerate thawing. When pea-sized ice crystals remain in the tube, remove it immediately and allow it to thaw at room temperature. Transfer the cryovials to a clean bench and slowly add the thawed cell suspension to 5 mL of hBMSCs complete culture medium. Gently pipette to mix evenly and centrifuge at 1000 rpm for 5 min. Discard the supernatant and add 2 mL of complete culture medium, gently pipetting to evenly disperse the cells into a single-cell suspension. Subsequently, seed the cells according to the culture dish specifications and add complete culture medium: 4 mL for 6 cm culture dishes and 8 mL for 10 cm culture dishes. Use the "cross-shading" method to horizontally shake the culture dishes to ensure even cell distribution. After confirming that the cells were evenly dispersed and without obvious cell clumps or fragments under an inverted microscope, the culture dish was transferred to a 37°C, 5% CO2 incubator for further culture.

[0046] (2) hBMSCs passage When hBMSCs reach 80%-90% confluence, passage them. Aspirate the old culture medium and slowly add 2 mL of sterile PBS buffer along the wall of the culture dish. Gently shake the dish to ensure thorough washing with PBS. Discard the washing buffer and repeat the PBS washing step once to completely remove residual culture medium. Slowly add 2 mL of 0.05% trypsin solution along the wall of the culture dish and gently shake to evenly cover the cell layer with the enzyme solution. When cell edges are observed to be wrinkled and gaps increased under a microscope, add 4 mL of complete culture medium to stop digestion (the culture medium volume should be at least twice the trypsin volume). Gently pipette the bottom of the culture dish until the adherent cells are completely detached. Centrifuge at 1000 rpm for 5 min. Discard the supernatant, add 2-3 mL of complete culture medium, and gently pipette to form a single-cell suspension. Seed the cells in new culture dishes at a ratio of 1:2 to 1:3, and add complete culture medium to the appropriate volume. Use the "cross-shading" method to evenly distribute the cells. After confirming that the cells were well dispersed and in normal condition by observing under an inverted microscope, the culture dish was placed back into a 37°C, 5% CO2 incubator for continued culture.

[0047] (3) cryopreservation of hBMSCs When hBMSCs reach 80%–90% confluence, cryopreservation can be chosen. Aspirate the old culture medium and slowly add 2 mL of sterile PBS buffer along the wall of the culture dish. Gently shake the dish to ensure thorough washing with PBS. Discard the washing buffer and repeat the PBS washing step once to completely remove residual culture medium. Slowly add 2 mL of 0.05% trypsin solution along the wall of the culture dish, gently shaking to evenly cover the cell layer with the enzyme solution. When cell edges are observed to be wrinkled and gaps increased under a microscope, add 4 mL of complete culture medium to stop digestion (the volume of the culture medium should be at least twice the volume of the trypsin). Gently pipette the bottom of the culture dish until the adherent cells are completely detached. Centrifuge at 1000 rpm for 5 min. Discard the supernatant and add 1 mL of cryopreservation solution, gently pipetting to prepare a single-cell suspension. Slowly inject the cell suspension into cryovials, avoiding air bubbles. Seal with sealing film and place in a -80°C freezer. Transfer to liquid nitrogen for cryopreservation the next day.

[0048] (4) hBMSCs recruitment experiment hBMSCs labeled with GFP using generation P2-P4 ( GFP+hBMSCs (human blastocyte mesenchymal stem cells) were used in experiments. These cells were transfected with lentivirus and stably expressed green fluorescent protein for cell adhesion, infiltration, and distribution tracking. The experiment consisted of three groups: T@GM, T@MGM, and T@MGM+S. The T@GM group used a GelMA hydrogel scaffold containing TGF-β3; the T@MGM group used a magnetically responsive GelMA hydrogel scaffold containing TGF-β3; and the T@MGM+S group used a magnetically responsive GelMA hydrogel scaffold containing TGF-β3, with an external static magnetic field applied (magnets were attached below the wells to provide a stable and uniform static magnetic field). Measurements showed that the magnetic induction intensity at the location of the magnetically responsive hydrogel scaffold within the wells was approximately 56.66 ± 0.36 mT, which is a weak magnetic field and meets the experimental requirements. Figure 8 ).

[0049] Remove the prepared hBMSCs cells from the incubator, aspirate the old culture medium, and slowly add 2 mL of sterile PBS buffer along the wall of the culture dish. Gently shake the dish to ensure thorough washing with PBS. Discard the washing buffer and repeat the PBS washing step once to completely remove residual culture medium. Slowly add 2 mL of 0.05% trypsin solution along the wall of the culture dish, gently shaking to evenly cover the cell layer with the enzyme solution. When cell edges are observed to be wrinkled and gaps increased under a microscope, add 4 mL of complete culture medium to stop digestion (the volume of the culture medium should be at least twice the volume of the trypsin). Gently pipette the bottom of the culture dish until the adherent cells are completely detached. Centrifuge at 1000 rpm for 5 min. Discard the supernatant, add an appropriate amount of complete culture medium, count the cells, and adjust the cell density to 2 × 10⁶ cells / mL. 6 Units / mL are available for use.

[0050] Sterilized hydrogel scaffold (GM and MGM) precursors were removed from a -20°C freezer and heated to a liquid state in a 37°C water bath. An appropriate amount of the hydrogel scaffold precursors was taken in a laminar flow hood, and TGF-β3 was added at a concentration of 0.1 μg / μl to form T@GM and T@MGM precursor gel solutions. These solutions were injected into a mold (1 cm inner diameter, 1.5 mm height) and photocrosslinked by irradiation with 405 nm light for 20 s, forming T@GM and T@MGM gel blocks. T@GM and T@MGM were placed in low-adhesion 24-well plates, and 1 mL of cell suspension was added, avoiding direct drop onto the T@MGM. 0.5 mL of culture medium was then added. For the T@MGM+S group, a magnet was attached directly below the wells with the N pole facing upwards. The plates were placed in a 37°C, 5% CO2 incubator for further culture, with the complete culture medium changed every 2-3 days. Samples were collected after 48 hours and 9 days, and the results are as follows. Figure 9 As shown.

[0051] Figure 9 The results showed that after two days of co-culturing, direct observation using a laser confocal microscope revealed [the presence of these features].GFP+ hBMSCs can spontaneously form spherical or sheet-like aggregates on the scaffold surface. Among them, the T@GM group cells are mainly spherical; the T@MGM group cell aggregates are also spherical, and show a tendency to gradually spread into sheets; while in the T@MGM+S group, although spherical aggregates can still be seen, the phenomenon of cells extending pseudopodia and spreading into sheets is more significant.

[0052] Observation of cross-sectional sections of the scaffold showed that after 2 days of co-culture, cells in all three groups mainly adhered to and aggregated on the scaffold surface, without significant infiltration and growth into the interior. By 9 days of co-culture, cells in all three groups had migrated to a certain depth into the hydrogel. Quantitative analysis showed that the migration depth of cells in the T@GM group was 127.7±23.93 μm, in the T@MGM group it was 131.6±38.73 μm, and in the T@MGM+S group it reached 256.9±45.38 μm. The migration distance of cells in the T@MGM+S group was significantly higher than that of the other two groups.

[0053] (5) hBMSCs chondrogenic induction experiment The experiments were conducted using P2-P4 generation hBMSCs. The experiments were divided into three groups: T@GM, T@MGM, and T@MGM+S. The T@GM group used a standard GelMA hydrogel scaffold containing TGF-β3; the T@MGM group used a magnetically responsive GelMA hydrogel scaffold containing TGF-β3 and MNPs; and the T@MGM+S group used a magnetically responsive GelMA hydrogel scaffold containing TGF-β3 and MNPs, with an external static magnetic field applied.

[0054] Remove the prepared hBMSCs cells from the incubator. In a clean bench, aspirate the old culture medium from the culture dish. Slowly add 2 mL of sterile PBS buffer along the wall of the dish, gently shaking to ensure thorough washing with PBS. Discard the washing solution and repeat the PBS washing step once to completely remove residual culture medium. Slowly add 2 mL of 0.05% trypsin solution along the wall of the dish, gently shaking to evenly cover the cell layer with the enzyme solution. When cell edges are observed to be wrinkled and gaps increased under an inverted microscope, immediately transfer the cells to a clean bench and add 4 mL of complete culture medium (the volume of the culture medium should be twice the volume of the trypsin) to stop trypsin digestion. Gently pipette the bottom of the culture dish with a 1000 μl pipette to completely detach the adherent cells. Transfer the cell suspension to centrifuge tubes and aliquot according to experimental needs after cell counting. Centrifuge at 1000 rpm for 5 min, then discard the supernatant in a clean bench, reserving the cell pellet for later use.

[0055] Sterilized hydrogel scaffold (GM and MGM) precursors were removed from a -20°C freezer and heated to a liquid state in a 37°C water bath. An appropriate amount of the hydrogel scaffold precursors was taken in a clean bench, and TGF-β3 was added at a concentration of 0.1 μg / μl to form T@GM and T@MGM precursor gel solutions. The cell pellet was resuspended in the T@GM and T@MGM precursor gel solutions, and the cells were repeatedly pipetted to ensure even distribution within the gel solution, maintaining a cell density of 2 × 10⁻⁶ cells / mL. 6 Cells / mL. The cell-containing gel was injected into a mold (1 cm inner diameter, 1.5 mm height), and photocrosslinked by irradiation with 405 nm light for 20 s, forming T@GM and T@MGM gel blocks. T@GM and T@MGM were placed in low-adhesion 24-well plates, and 1.5 mL of complete culture medium was added. For the T@MGM+S group, a magnet was attached directly below the well plate with the N pole facing upwards to provide axial micromechanical stimulation to the cells within the hydrogel through magnetic attraction. After the operation, the well plates were placed in a 37℃, 5% CO2 incubator for further culture. One day later, the culture medium was changed to chondrogenic induction medium, and then changed every 2-3 days. After 16 days of chondrogenic induction culture, samples were harvested, and the results are as follows. Figure 10 As shown.

[0056] (6) Immunofluorescence staining (SOX9, ACAN, COL2A1) Remove frozen sections from the -20℃ freezer and allow them to reach room temperature for 30 minutes. Wash three times with PBS, 5 minutes each time. Circle the tissue with a histochemical pen, cover the tissue with strong immunofluorescence permeation buffer, and permeate at room temperature for 15 minutes. Wash three times with PBS, 5 minutes each time. Block with immunofluorescence staining blocking solution at 37℃ for 30 minutes to 1 hour, and wash once with PBS. Prepare antibody dilution buffer (SOX9, ACAN, COL2A1) at a ratio of 1:100, add it to the tissue, and incubate overnight at 4℃. Wash three times with PBS, 5 minutes each time. Prepare the corresponding secondary antibody dilution buffer at a ratio of 1:200, add it to the tissue, and incubate at 37℃ for 1 hour. Wash three times with PBS, 5 minutes each time. Mount the slides with mounting buffer containing DAPI to quench fluorescence, observe and photograph under a fluorescence microscope. The results are as follows. Figure 10 As shown.

[0057] Figure 10Immunofluorescence staining results of key chondrogenic differentiation markers SOX9, COL2A1, and ACAN after 16 days of chondrogenic induction culture showed that some cells in all three groups were positively stained. SOX9, a core transcription factor for chondrogenic differentiation, was mainly expressed in the nucleus of induced cells, while COL2A1 and ACAN were mainly distributed in the periphery and extracellular matrix, exhibiting characteristic positive red fluorescence signals. Statistical quantitative analysis showed that the positive cell rates of SOX9, COL2A1, and ACAN in the T@MGM+S group were significantly higher than those in the T@GM and T@MGM groups.

[0058] The above results indicate that under the intervention of static magnetic field combined with magnetic response growth plate repair scaffold, hBMSCs can be more effectively promoted to differentiate into chondrocytes, showing better chondrogenic induction efficiency.

[0059] Experimental Example 3 Experiment 3 of this invention conducted an in vivo application experiment of the magnetically responsive growth plate repair scaffold. The specific steps are as follows: Construction of a rabbit model of supercritical size marginal tibial growth plate defect The magnetically responsive scaffold was prepared according to Example 1. A supercritical-sized marginal growth plate defect model was constructed on the medial side of the rabbit tibia, with a defect size of 5 mm in length, 5 mm in depth, and 2 mm in width. Currently, clinical observations suggest that over 30% of injuries have extremely poor prognoses, and this defect size far exceeds the critical size.

[0060] The animal experiments were divided into 4 groups: The Blank group, serving as a blank control, involved only creating a lesion model on the animal's growth plate without filling it with any implants.

[0061] In the T@GM group, after lesions were created in the animal growth plates, T@GM hydrogel scaffolds containing TGF-β3 were filled.

[0062] In the T@MGM group, after simulating damage to animal growth plates, a T@MGM magnetic response growth plate repair scaffold containing TGF-β3 and MNPs was filled.

[0063] In the T@MGM+S group, after creating a damage model of the animal growth plate, a T@MGM magnetic response growth plate repair scaffold was filled in. At the same time, a magnetic sheet was implanted parallel to the metaphysis at a distance of 2 mm from the growth plate, thereby providing a uniform and stable static magnetic field locally and giving the cells in the magnetic response growth plate repair scaffold axial micromechanical stimulation.

[0064] 1) Preoperative preparation Animal preparation: Purchase 4-week-old male rabbits and acclimate them in the animal facility for 2 to 6 weeks to ensure they are healthy and in good condition.

[0065] Preparation of materials: All surgical instruments should be autoclaved in advance, and implants should be sterilized in a suitable manner. Preheat the GM and MGM hydrogel precursors to 37°C in a water bath and encapsulate TGF-β3 to form T@GM and T@MGM precursor solutions for later use.

[0066] Personnel preparation: All personnel involved in animal experiments wore disposable surgical gowns, and the chief surgeon and assistant scrubbed their hands and wore surgical gloves according to the surgical procedure.

[0067] 2) Surgical procedures Six-week-old male rabbits (weighing 1.2 ± 0.1 kg) were anesthetized via intravenous injection of 2% sodium pentobarbital in the ear, with the dosage calculated at 1 mL / kg. After the rabbit's corneal reflex disappeared, the surgical area was prepared. The surgical area was identified and marked using surface landmarks. Using the area near the proximal tibia of the right hind limb as the center, the hair within a 5 cm radius of the surgical area was thoroughly removed to fully expose the skin. The prepared area was disinfected three times from the inside out with iodine-soaked cotton balls. Following surgical principles, the area was draped, exposing only the skin of the surgical site. Local anesthesia was achieved by subcutaneous injection of lidocaine, creating a wheal approximately 1 cm in diameter. The skin was incised, and the subcutaneous tissue was bluntly dissected. Hemostasis was achieved by applying pressure to the severed blood vessels using hemostatic forceps. The strip ligament near the proximal tibia of the knee joint was fixed with a retractor, fully exposing the medial growth plate of the proximal tibia of the right hind limb. Guided by a steel ruler, a pointed scalpel was used to delineate the area to be destroyed around the growth plate, ensuring a consistent damage extent. A marginal defect model of the growth plate was constructed, and the growth plate was violently destroyed using a dental burr, forming a 5 mm long, 5 mm deep, and 2 mm wide osteochondral defect. Hydrogel precursor solution was aspirated and injected into the damaged area of ​​the growth plate, and immediately irradiated with 405 nm light to induce in-situ photocrosslinking, completing the implantation of the hydrogel scaffold. For the T@MGM+S group, an additional 5 mm long, 5 mm deep, and 1.2 mm wide lesion strip was created parallel to the metaphysis 2 mm from the lesion area using a dental burr, and a magnetic sheet was implanted to provide a uniform and stable static magnetic field (e.g., ...). Figure 11 (As shown). The deep fascia, superficial fascia, and skin were sutured layer by layer, and penicillin was injected intramuscularly into the right rump of the rabbit at a dose of 20,000 U / kg (for 3 consecutive days from the day of surgery; if infection occurs, double the usual dose). After the rabbit recovered, it was transferred to the animal room for regular feeding.

[0068] 3) Postoperative tissue sampling Samples were taken on the day of surgery and at 2 and 8 weeks post-surgery. On the day of surgery, only 3 rabbits were sampled to assess the accuracy of the model and the proportion of the lesion area; the specific procedures were the same as at 2 and 8 weeks post-surgery.

[0069] Two weeks after surgery, 3 rabbits were randomly selected from each group, anesthetized via ear marginal vein injection with 2% sodium pentobarbital at a dosage of 1 mL / kg. After confirming the absence of corneal reflex in the rabbits, air was injected through the ear marginal vein to euthanize the animals via air embolism.

[0070] After euthanasia, the osteochondral tissue in the injured area was directly excised with a pointed blade at the modeling site of the proximal tibial growth plate of the right hind limb, with a volume of approximately 125 mm 3 (5 mm in length, 5 mm in width, 5 mm in depth). Three of the samples were quickly rinsed with PBS and then immersed in electron microscope fixative for subsequent SEM observation.

[0071] Bilateral tibias were harvested from the remaining rabbits at 2 weeks post-operation and the rabbits at 8 weeks post-operation, the soft tissues on them were removed, and the tibias were immersed in 4% paraformaldehyde as soon as possible for fixation for 3 days.

[0072] The tibias were taken out, rinsed with tap water for 10 minutes, then transferred to a rapid decalcification solution for decalcification. The decalcification solution was replaced every 2-3 days, and the tissue was trimmed into thick slices during this period. The completion of decalcification was marked by the fact that a 1 mL syringe needle could be easily inserted into the tissue.

[0073] The decalcified samples were taken and rinsed with tap water for 30 minutes to completely remove the residual decalcification solution. Subsequently, the samples were placed in a dehydrator and dehydrated for 24 hours according to the preset program.

[0074] After dehydration, paraffin embedding was performed, followed by sectioning for preservation.

[0075] Additional tissue samples of the heart, liver and kidney were harvested from the rabbits at 8 weeks post-operation to evaluate the in vivo safety of the implant in animals. After being fixed with 4% paraformaldehyde for 3 days, the tissues were subjected to dehydration with sucrose solution, OCT embedding, and frozen sectioning.

[0076] 4) Evaluation of postoperative repair effect A. X-ray analysis The fixed and undecalcified tibial samples were taken for imaging detection using a medical X-ray radiography system to capture anteroposterior X-ray films. The image analysis software supporting the radiography system was used to measure the length of bilateral tibias and the Medial Proximal Tibial Angle (MPTA).

[0077] B. MicroCT analysis The fixed and undecalcified tibial samples were radiographed using a dedicated small-animal micro computed tomography system (Micro-CT, instrument model: SkyScan 1276), with the resolution set to 20 μm, scanning voltage of 70 kV, and current of 200 μA.

[0078] DataViewer software was used for basic image observation and saving. The images were rotated and adjusted according to standard rotations to ensure the tibial sample was in an upright position in the coronal, sagittal, and cross-sectional views for subsequent analysis; the images were then saved again. For samples taken on the day of surgery, the software allowed for the measurement of damage extent data in a two-dimensional plane.

[0079] Using CTAn software, the damaged areas of the growth plate were delineated layer by layer and marked as regions of interest, with a thickness of 3 mm. This allowed for the analysis of indicators such as BMD, Bone Volume, and Tb.N within these areas.

[0080] CTvox is used to perform visualization, 3D reconstruction of the scanned image, and obtain a 3D image.

[0081] C. Histological staining HE staining: Dewax and rehydrate the sections in each group. Immerse the sections in hematoxylin staining solution for 3 min, then gently rinse with running water for 1 min; immerse the sections in 1% hydrochloric acid ethanol solution for 2 s, then immerse in tap water for 1 min; immerse the sections in ammonia solution for 1 min, then immerse in tap water for 1 min; immerse the sections in eosin staining solution for 2 min, then immerse in tap water for 1 min to complete the staining; immerse the sections in 95% and 100% ethanol for 1 min each to remove water from the tissue. Finally, place the sections in xylene (clearing solution) in a fume hood for 30 s. After removing the sections, air dry them and mount them with neutral resin.

[0082] D. Safranin O-Fix Green Staining: Dewax and rehydrate all sections. Before staining, mix Weigert stain solution A and solution B in a 1:1 volume ratio, preparing fresh before use. Add approximately 100 μl of the freshly prepared Weigert stain solution to each section and stain at room temperature for 5 min. Discard the stain solution, add acidic differentiation solution for 5 s, and immediately rinse the sections quickly in a first tub of purified water, then transfer to a second tub of purified water for a thorough rinse for 10 min. After rinsing, add Fast Green stain solution and stain for 10 min. Treat with weakly acidic differentiation solution for 8 s to remove excess Fast Green, then add Safranin O stain solution for 15 min. Discard the Safranin O stain solution and rinse the sections with 95% ethanol to remove excess stain. Immerse the sections sequentially in 95% and 100% ethanol for 1 min each to remove moisture from the tissue. Finally, place the sections in xylene (clearing solution) in a fume hood for 30 s. After removing the slices, let them air dry and then seal them with neutral resin.

[0083] E. Immunofluorescence staining analysis Dewaxing and rehydrating of all slides were performed. Citrate was used for COL2A1 staining repair. The acid repair conditions were: sodium citrate (pH 6.0) in an 85°C water bath for 10 min, followed by natural return to room temperature. After repair, the slides were washed three times with PBS buffer, 5 min each time. The tissue samples to be stained were traced with an immunohistochemical pen, and immunostaining blocking solution was added to completely cover the slides. The slides were incubated at 37°C for 1 hour. COL2A1 primary antibody was diluted 1:100 with immunostaining primary antibody dilution buffer. The diluted primary antibody was added to completely cover the slides. The slides were incubated overnight at 4°C. The next day, the slides were washed three times with PBS buffer, 5 min each time. Cy3 secondary antibody was diluted 1:200 with immunostaining secondary antibody dilution buffer. The diluted secondary antibody was added in the dark to completely cover the slides. The slides were incubated at 37°C for 1 hour. The slides were then washed three times with PBS buffer, 5 min each time. Gently wipe the slide dry and mount it with a DAPI-containing anti-fluorescence quenching mounting solution. Observe and photograph it under an inverted fluorescence microscope as soon as possible.

[0084] Figure 12 The study showed that three rabbits were euthanized and their tissues were collected after modeling on the day of surgery. X-rays were taken, and the lengths of the left and right tibias and MPTA of each rabbit were measured. The results showed no significant difference in the lengths of the left and right tibias and MPTA of the rabbits, which can be used as autocontrols for subsequent analysis.

[0085] Simultaneously, the accuracy of the surgical site was verified using Micro-CT, and the proportion of growth plate damage area was calculated. The area circled in red solid circle represents residual growth plate tissue after surgery, the area circled in yellow dashed circle represents damaged or missing growth plate tissue, and the area circled in white dashed circle represents the magnetic implantation site. Multi-level statistical calculations showed that the growth plate damage area accounted for approximately 41.63% ± 7.92%. Current clinical practice generally considers a damage area exceeding 30% to have an extremely poor prognosis, inevitably leading to severe deformities. The damage model used in this invention closely matches clinical practice and its extent far exceeds the critical size, making it a pioneering achievement in the field.

[0086] To further verify the cell recruitment capacity of each repair system in animals, SEM was used to observe the local micromorphology of the damaged area, including the implant. Figure 13 SEM observation of the damaged area two weeks after modeling showed that only osteocytes (green pseudo-color) were visible in the Blank group. However, spindle-shaped stem cell-like cells (red pseudo-color) were clearly observed in the hydrogel-tissue interface area of ​​the T@GM, T@MGM, and T@MGM+S groups, confirming that the scaffolds in each group had the bioactivity to actively recruit stem cells.

[0087] Figure 14Two weeks after modeling, HE staining panoramic images visually presented the macroscopic repair status of the proximal tibial growth plate defect area. Region i in the image marks the defect location, with a high-magnification image displayed below. Results showed that the Blank group consisted of numerous trabeculae and interwoven collagen fibers, indicating premature bone replacement in the defect area. In contrast, residual hydrogel scaffolds were visible in the T@GM, T@MGM, and T@MGM+S groups, with good integration between the scaffolds and surrounding host tissues. Specifically, the T@GM group had a larger residual scaffold area, mainly distributed in the central region of the defect, with only scattered cell attachments within; the T@MGM group had a smaller residual scaffold area, adjacent to the cortical bone, with a small number of cells also visible at the tissue interface; while the T@MGM+S group's scaffold achieved deep integration with the surrounding tissue, with a large number of densely distributed cells both inside and at the boundaries.

[0088] Safranin O-Fixed Green staining further revealed the cellular and matrix characteristics of the defect area. In the Blank group, mature osteocytes were abundant within the trabeculae, with no signs of chondrogenic tissue. In the T@GM group, cells of varying morphology were observed, including chondrocyte-like cells, but no obvious pale red matrix deposition was seen around the cells. This indicates that although stem cells may be induced to differentiate into chondrocyte progenitors, they failed to secrete large amounts of chondrogenic ECM, thus limiting the positive feedback of chondrogenic differentiation. The T@MGM group was similar to the T@GM group, with a small number of chondrocyte-like cells. In contrast, the T@MGM+S group scaffold was not only filled with chondrocyte-like cells but also accompanied by a large accumulation of collagen fibers, forming clearly defined new cartilage tissue; simultaneously, typical signs of endochondral ossification were observed around the residual scaffold. This confirms that in the early stages of injury, the T@MGM+S composite material can effectively recruit endogenous stem cells in situ and induce their chondrogenic differentiation.

[0089] 5) Assessment of growth potential recovery in damaged areas of the growth plate Three time points were selected: the day of modeling, 2 weeks post-operation, and 8 weeks post-operation. X-ray images were integrated to measure the distance from the implanted magnetic patch to the growth plate damage area in the T@MGM+S group, which was named magnetic-epithelial distance. Bilateral tibial length and MPTA were measured. The recovery effect of growth potential in the defect area was visually assessed through changes in magnetic-epithelial distance.

[0090] The following formulas are used to calculate the shortening of the operated limb and the angle of angular deformity:

[0091] In the formula: δL — Tibial shortening amount / mm; L 1—Length of the healthy side tibia / mm; L 2——Tibia length on the operated side / mm.

[0092]

[0093] In the formula: θ —Abnormal angle / °; α 1—Healthy side MPTA / °; α 2——Operative side MPTA / °.

[0094] Eight weeks after modeling, the tibial length shortening (δL) in each group was: Blank group 6.10±0.93 mm; T@GM group 5.01±1.17 mm; T@MGM group 3.72±0.46 mm; T@MGM+S group 4.13±0.89 mm. For angular deformity (θ), the θ was 36.23±2.05° in the Blank group; 33.35±3.26° in the T@GM group; 24.31±4.25° in the T@MGM group; and 24.63±2.66° in the T@MGM+S group.

[0095] Figure 15 The results showed that, through statistical analysis, in a rabbit model of proximal tibial growth plate defects, the long-term evaluation results at 8 weeks post-surgery indicated that T@GM could not effectively prevent limb shortening and angular deformity, while both T@MGM and T@MGM+S programs could effectively improve lower limb shortening and angular deformity, and the two programs had comparable corrective effects on deformity.

[0096] To further and more intuitively observe the repair of the damaged area and the overall morphology of bone bridge formation 8 weeks after modeling a proximal tibial growth plate defect in rabbits, Micro-CT three-dimensional reconstruction and visualization analysis were performed on the samples, such as... Figure 16 As shown. At the same time, by accurately delineating and reconstructing the damaged area (red), the spatial distribution, morphological structure and continuity of the trabeculae and newly formed bone bridges in each group of damaged areas are magnified and displayed, which intuitively reflects the overall effect of bone repair in each group from a qualitative perspective.

[0097] Figure 16 The results showed that the Blank group had formed a dense plate-like bone bridge, which severely hindered further growth of the medial tibia; the bone bridges in the T@GM group, T@MGM group and T@MGM+S group gradually decreased, which directly demonstrated the inhibitory effect of each repair system on the formation of bone bridges.

[0098] Based on this, Micro-CT was used to perform quantitative statistical analysis on the relevant parameters of bone bridges in the damaged area, focusing on three indicators: bone mineral density (BMD), bone volume fraction (BV), and trabecular number (Tb.N). This objectively evaluated the differences in new bone formation and mineralization in the damaged areas of each group, thereby comprehensively and systematically evaluating the prevention and treatment effect of bone bridge formation.

[0099] Figure 16 The BV statistics showed that the Blank group had a BV of 1.56 ± 0.50 mm. 3 The T@GM group measured 1.10 ± 0.26 mm. 3 The T@MGM group measured 1.05 ± 0.27 mm. 3 The T@MGM+S group was 0.48±0.18 mm. 3 The Tb.N statistical results showed that: the Blank group was 2.76±0.34 1 / mm; the T@GM group was 1.84±0.30 1 / mm; the T@MGM group was 1.48±0.28 1 / mm; and the T@MGM+S group was 1.31±0.55 1 / mm. The BMD statistical results showed that: the Blank group was 0.31±0.035 g / cm³. 3 The T@GM group was 0.23 ± 0.031 g / cm³. 3 The T@MGM group was 0.20 ± 0.026 g / cm³. 3 The T@MGM+S group was 0.14±0.060 g / cm³. 3 Statistical analysis showed that the bone volume (BV) of the T@MGM+S group was significantly lower than that of the other three groups, directly demonstrating that this group could minimize the formation of bone bridges. Simultaneously, this group effectively reduced the density of bone bridges and decreased the degree of local ossification. Therefore, the T@MGM combined with a static magnetic field repair strategy can significantly reduce the density and volume of bone bridges in the growth plate damage area, effectively preventing bone bridge formation, and is the optimal intervention method.

[0100] 6) The protective effect of the magnetically responsive growth plate repair scaffold on the cartilage tissue of the growth plate in the vicinity of injury. The height of the growth plate adjacent to the injury zone was measured in each group of samples 8 weeks post-surgery. Figure 17 As shown.

[0101] Figure 17 The results showed that the growth plate height in the Blank group was 328.5±7.22 μm; in the T@GM group, it was 294.0±34.50 μm; in the T@MGM group, it was 302.6±44.05 μm; and in the T@MGM+S group, it was 510.1±13.04 μm. The growth plate height in the lesion-adjacent area of ​​the T@MGM+S group was significantly higher than that of the other three groups, indicating that the magnetically responsive growth plate repair scaffold had a significant protective effect on the growth plate tissue in the lesion-adjacent area under a static magnetic field.

[0102] 7) The effect of magnetically responsive growth plate repair scaffolds on the growth potential of damaged growth plates. The T@MGM+S repair system can effectively inhibit bone bridge formation and promote cartilage regeneration, while also protecting the growth plate tissue in the adjacent area of ​​the injury. Three time points were selected: the day of modeling, 2 weeks post-surgery, and 8 weeks post-surgery. Imaging data were integrated, and the recovery effect of the growth potential of the defect area was visually assessed through changes in magnetic-epithelial distance (MGPD). Figure 18 The X-ray images of the bilateral tibias of rabbits at three time points are presented. The red lines in the figures represent the magnetic-epithelial distance (MGPD) measurement markers. Statistical analysis showed that the MGPD on the day of modeling was 1.79±0.50 mm, which was basically consistent with the pre-set standard for surgical modeling. Two weeks postoperatively, the MGPD increased to 6.49±0.37 mm, and further increased to 10.58±0.52 mm at eight weeks postoperatively. Significant statistical differences were found in pairwise comparisons at all three time points, and the MGPD showed a significant increasing trend with postoperative time. These results indicate that the T@MGM+S repair system can stably maintain its functional reconstruction effect on proximal tibial growth plate defects in rabbits up to eight weeks postoperatively, demonstrating a long-term repair advantage.

[0103] 8) Safety evaluation of implants Heart, liver, and kidney tissues were collected from rabbits in each group 8 weeks after modeling, frozen sections were prepared, and HE staining and histological observation were performed.

[0104] like Figure 19 As shown, the myocardial tissue of rabbits in each group was neatly arranged, with regular myocardial fiber morphology and intact structure, and no obvious degeneration, necrosis or inflammatory cell infiltration was observed; the liver tissue showed clear lobular structure and regular hepatocyte arrangement, and no hepatocyte degeneration, necrosis or obvious inflammatory reaction was observed; the kidney tissue showed intact and clear glomerular structure and orderly arrangement of renal tubules, and no obvious atrophy, dilation or inflammatory infiltration or other pathological changes were observed.

[0105] Compared with the blank control group, no obvious cardiotoxicity, liver toxicity or kidney toxicity was observed in the heart, liver and kidney tissues of each experimental group, and no abnormal pathological changes were observed in the structure of important organs and tissues. This suggests that the implanted materials used in different repair strategies in this study have no obvious organ toxicity side effects on rabbits and have good in vivo biocompatibility.

[0106] Experimental data are expressed as mean ± standard deviation. The t-test was used to compare differences between two groups, and one-way ANOVA was used for pairwise comparisons among multiple groups. All statistical calculations and graphical visualizations were performed using the professional analysis software GraphPad Prism 10.1.2. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns indicates no statistical difference. Each experimental group contained at least 3 independent samples (i.e., n ≥ 3 per group).

[0107] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A magnetically responsive growth plate repair scaffold, characterized in that, The raw materials for preparing the magnetic response growth plate repair scaffold include 80-120 parts of dry GelMA, 4-6 parts of MNPs, and 0.05-0.2 parts of TGF-β3.

2. The magnetically responsive growth plate repair scaffold according to claim 1, characterized in that, The raw materials for preparing the magnetic response growth plate repair scaffold include 100 parts of dry GelMA, 5 parts of MNPs, and 0.1 parts of TGF-β3.

3. A method for preparing the magnetically responsive growth plate repair scaffold according to claim 1, characterized in that, Includes the following steps: (1) Dissolve the dry GelMA and heat and stir to obtain a GelMA hydrogel solution; (2) Dissolve MNPs in the GelMA hydrogel solution of step (1) to obtain a magnetically responsive GelMA solution; (3) TGF-β3 was added to the magnetically responsive GelMA solution to obtain the precursor of the magnetically responsive growth plate repair scaffold; (4) Take the precursor of the magnetic response growth plate repair scaffold, heat it to liquid state, and photocrosslink it to obtain the magnetic response growth plate repair scaffold.

4. The preparation method according to claim 3, characterized in that, In step (1), the temperature of the heating and stirring is 35~45℃.

5. The preparation method according to claim 3, characterized in that, In step (4), the heating temperature is 35~38℃.

6. The preparation method according to claim 3, characterized in that, In step (4), the conditions for photocrosslinking are: irradiation with 405nm light for 20~40 s.

7. The application of a magnetically responsive growth plate repair scaffold as described in any one of claims 1 to 2 or a magnetically responsive growth plate repair scaffold prepared by the preparation method described in any one of claims 3 to 6 in the repair of growth plates and the preparation of growth plate repair scaffolds.

8. A method for evaluating the growth potential of a magnetically responsive growth plate repair scaffold, characterized in that, Includes the following steps: (1) The degree of limb shortening and angular deformity is detected by calculating the amount of limb shortening and the angle of angular deformity on the operated side. The formula used is as follows: The amount of limb shortening on the operated side: In the formula: δL — Tibial shortening amount / mm; L 1—Length of the healthy side tibia / mm; L 2—Tibia length on the operated side / mm; Angle of angular deformity: In the formula: θ —Abnormal angle / °; α 1—Healthy side MPTA / °; α 2—Operative side MPTA / °; (2) The recovery effect of growth potential in the defect area can be intuitively evaluated by the change of magnetic-epistem distance.

Images