A developmental growth plate lamellar scaffold and methods of making and using the same

Abstract

The application provides a developmental growth plate layered scaffold and a preparation method and application thereof, and belongs to the technical field of bone tissue engineering materials. The developmental growth plate layered scaffold provided by the application is divided into three layers, the upper layer is a cartilage cell layer, the middle layer is a blood vessel barrier layer, and the lower layer is a bone formation layer. The blood vessel barrier layer can be used as biological glue to bond the upper layer and the lower layer, so that the composite scaffold structure is stable and is not easy to break; and the upper layer contains bone marrow mesenchymal stem cells (BMSCs), and the middle layer and the lower layer are cell-free. Under the in-vitro chondrogenic induction culture condition, the BMSCs in the upper layer can pass through the middle layer and colonize in the lower layer, and in the process, part of the BMSCs can differentiate into cartilage cells. The middle layer containing thrombospondin 1 (TSP1) has specific intervention effect on cell migration, which is manifested as inhibition of the migration of vascular endothelial cells, but has no effect on the migration of BMSCs.

Description

Technical Field

[0001] This invention relates to the field of bone tissue engineering materials technology, and in particular to a developmental growth plate layered scaffold, its preparation method, and its application. Background Technology

[0002] The growth plate, also known as the epiphyseal plate, is a wavy, specialized cartilage tissue located between the secondary ossification center and the metaphysis, driving bone growth. Epiphyseal plate chondrocytes are mechanically fragile and highly susceptible to physical damage. Due to their limited regenerative capacity, they are easily replaced by bone tissue during repair, creating "bone bridges." This localized growth arrest can lead to serious complications such as angular deformities and limb length discrepancies. Currently, the commonly used clinical treatment is bone bridge resection combined with inert material filling, but the repair effect is unsatisfactory because the filling material lacks biomimetic properties and is difficult to adapt to the damaged microenvironment. Therefore, there is an urgent need to develop a biomimetic scaffold to restore the developmental potential of damaged growth plates.

[0003] As is well known, the growth plate is a heterogeneous and dynamically changing developmental tissue. When constructing alternative grafts for the growth plate, its developmental biology characteristics should be considered: First, the resting zone of the growth plate serves as a reservoir for skeletal stem cells, supplying chondrocytes to the proliferation and hypertrophy zones. These chondrocytes further hypertrophy or transdifferentiate to participate in osteoogenesis, and this dynamic cellular order drives bone growth. Second, the main matrix of the growth plate is collagen and glycosaminoglycans. Calcium salt deposits, such as hydroxyapatite, gradually appear in the hypertrophy and mineralization zones. Furthermore, the matrix porosity and reduced modulus gradually increase from the resting zone to the mineralization zone, and this heterogeneous structure signifies the transformation of cartilage into bone. Third, growth plate chondrocytes exist in an avascular and nerveless microenvironment because of the blood supply barriers at the epiphysis and metaphysis, which maintain the proliferative phenotype of chondrocytes. Growth plate damage leads to disruption of the blood supply barrier, causing chondrocytes to hypertrophy rapidly in an oxygen-rich microenvironment, resulting in bone bridge formation through endochondral ossification. Simultaneously, blood infiltration inevitably leads to blood vessels growing into areas that were originally without blood supply, allowing stem cells to form bone bridges through intramembrane ossification.

[0004] In summary, an ideal biomimetic scaffold needs to mimic biomimetic composition and heterogeneous structure. Specifically, it needs to simulate the different distributions of collagen matrix and minerals in each layer of the growth plate, the changes in porosity and mechanical properties, and the reconstruction of the blood supply barrier. Summary of the Invention

[0005] The purpose of this invention is to provide a developmental growth plate layered scaffold, its preparation method and application, wherein the developmental growth plate layered scaffold can simulate and reproduce the developmental and functional characteristics of the growth plate.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a developmental growth plate layered scaffold comprising upper, middle and lower layers; the upper layer is a chondrocyte layer, the middle layer is a vascular barrier layer, and the lower layer is an ossification layer;

[0008] The chondrocyte layer is loaded with bone marrow mesenchymal stem cells;

[0009] The vascular barrier layer is loaded with platelet-reactive protein 1;

[0010] The ossified layer is composed of type I collagen and hydroxyapatite.

[0011] Preferably, the method for preparing the chondrocyte layer includes the following steps:

[0012] Bone marrow mesenchymal stem cells with a cell fusion rate of 80-90% were digested and resuspended to obtain a cell resuspension. The cell resuspension was dropped onto a microcarrier, allowed to stand, and induced differentiation medium was added for dispersion treatment. The induced differentiation medium was replaced and incubated to obtain the chondrocyte layer.

[0013] More preferably, the density of the cell resuspension is 1–3 × 10⁻⁶. 6 The concentration of cells / mL is 37°C, the standing temperature is 2-3 hours, the standing time is 2-3 days, the frequency of changing the induction differentiation medium is 2-3 times per day, and the incubation time is 5-7 days.

[0014] Preferably, the method for preparing the vascular barrier layer includes the following steps:

[0015] Methacrylamide gelatin and a photoinitiator were dissolved separately, and platelet-reactive protein 1 was added. The mixture was then irradiated with blue light or ultraviolet light to obtain the vascular barrier layer.

[0016] More preferably, the dissolution is performed using a phosphate buffer solution, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine, and the effective concentration of platelet-reactive protein 1 is 5 μg / mL.

[0017] Preferably, the method for preparing the ossification layer includes the following steps:

[0018] Type I collagen was dissolved and hydroxyapatite was added to obtain 3D printing bio-ink. The ink was then added to a 3D bioprinter for printing to obtain a type I collagen / hydroxyapatite scaffold. The type I collagen / hydroxyapatite scaffold was freeze-dried and cross-linked to obtain the ossification layer.

[0019] More preferably, acetic acid is used for dissolution; the mass ratio of type I collagen to hydroxyapatite is 1 to 2:1; the freeze-drying time is 4 to 6 hours; and genipin is used for cross-linking.

[0020] The present invention also provides a method for assembling the aforementioned developmental growth plate layered scaffold, comprising the following steps:

[0021] The chondrocyte layer was cut into 5×5mm pieces and adhered to the vascular barrier layer to obtain a pre-assembled scaffold. A polyvinylidene fluoride membrane was inserted from one side of the pre-assembled scaffold, and the insertion was stopped at 1 / 2 of the pre-assembled scaffold. The pre-assembled scaffold was then lifted and pushed onto the ossified layer for cross-linking. It was then transferred to a chondrogenic induction culture medium for culture to obtain the developmental growth plate layered scaffold.

[0022] Preferably, the crosslinking is performed under a 405nm light source; the crosslinking time is 14–16 s; the culture temperature is 37°C; and the culture time is 5–7 days.

[0023] The present invention also provides the application of the aforementioned developmental growth plate layered scaffold in the preparation of products for repairing epiphyseal injuries.

[0024] The beneficial effects of this invention compared to the prior art are as follows:

[0025] The developmental growth plate layered scaffold prepared in this invention consists of three layers: an upper chondrocyte layer, a middle vascular barrier layer, and a lower ossification layer. The vascular barrier layer acts as a bio-adhesive to bond the upper and lower layers, stabilizing the composite scaffold structure and preventing breakage. The upper layer of the developmental growth plate layered scaffold contains bone marrow mesenchymal stem cells (BMSCs), while the middle and lower layers are cell-free. Under in vitro chondrogenic induction culture conditions, the BMSCs in the upper layer can penetrate the middle layer and colonize the lower layer. During this process, some BMSCs can differentiate into chondrocytes. The middle layer, containing TSP1, has a specific intervention effect on cell migration, inhibiting the migration of vascular endothelial cells but having no effect on the migration of BMSCs. Compared to traditional scaffolds, this invention has the function of inhibiting angiogenesis and supports the migration and differentiation of BMSCs, ultimately reconstructing the dynamic evolutionary order of cells in vitro, similar to natural growth plate cells.

[0026] Experiments show that implanting the developmental growth plate layered scaffold prepared according to this invention into the growth plate defect site effectively inhibits angiogenesis in that area, reduces the number of bone bridges, and ultimately significantly alleviates angular and shortening deformities of the damaged tibia. This invention has achieved success in the repair of severe marginal growth plate defects, demonstrating better efficacy and a wider range of applications compared to traditional scaffolds. Furthermore, the local delivery system of anti-angiogenic factors encapsulated within the scaffold significantly avoids side effects on other healthy bones compared to traditional systemic interventions.

[0027] This invention relates to a developmental growth plate layered scaffold with a layered structure, prepared based on a thorough understanding of the physicochemical and biological properties of growth plates. This developmental growth plate layered scaffold is similar to a natural growth plate in terms of matrix composition, heterogeneous structure, mechanical properties, and cell state. Furthermore, cells in the upper layer of the scaffold can penetrate the middle layer and colonize in the lower layer. Compared to traditional growth plate replacement scaffolds, the layered scaffold prepared in this invention has homologous matrix components and good biocompatibility, supporting cell adhesion and migration. Compared to traditional single-layer or double-layer scaffolds, this invention introduces a TSP1-mediated anti-angiogenic layer between the cartilage layer and the ossified layer for the first time, endowing the scaffold with strong anti-angiogenic potential.

[0028] Compared with traditional technologies, the developmental growth plate layered scaffold provided by this invention has breakthrough improvements in the following aspects:

[0029] ① In terms of construction strategy, the three-layer developmental growth plate scaffold proposed in this invention is more in line with the physiological heterogeneity characteristics compared to traditional single-layer and double-layer scaffolds. In particular, the addition of the vascular barrier layer effectively blocks vascular ingrowth and blood infiltration, which is of great significance for inhibiting osteogenic formation and maintaining cartilage phenotype. In addition, this invention only seeded stem cells in the upper layer, confirming that these cells can penetrate the middle hydrogel layer and colonize in the lower layer, and that both differentiated chondrocytes and undifferentiated stem cells coexist, thus preliminarily reproducing the dynamic evolution of natural growth plate cells.

[0030] ② Regarding the selection of substrates, the upper layer of this invention is gelatin microspheres, the middle layer is methacrylamide gelatin (GelMA), and the lower layer is collagen and hydroxyapatite. The three layers are homologous and easily integrated, thereby ensuring that when cells migrate from the upper layer to the lower layer, they will not detach, hinder migration, or even die due to differences in the peripheral matrix.

[0031] ③ In terms of manufacturing technology, the upper layer uses a static culture system to fuse MSC microspheres into one, which is essentially a cell-secreted matrix interaction and self-assembly, greatly helping cells adapt to the pathological microenvironment; the middle layer, GelMA, is a photocurable hydrogel that can uniformly load TSP1 and act as an adhesive to bond the upper and lower layers, and its large-pore structure facilitates cell migration and penetration; the lower layer is prepared using 3D printing technology to ensure controllable pores and nutrient transport. In summary, this layered manufacturing and reassembly technology simulates the porosity and gradual mechanical properties of natural growth plates.

[0032] ④ Regarding evaluation methods, traditional repair strategies often employ central or small-area marginal epiphyseal plate defect models in rats or rabbits. These models are simple to prepare and relatively easy to repair. This invention selects young white rabbits as the research subject, creating a large-area growth plate defect (5mm wide, 5mm deep, and 3mm high) on the medial side of the proximal tibia. This model is a typical marginal defect, and the size of the damaged area is rare in the field. The highly complex damage model created in this invention is of great significance for verifying the effectiveness of layered scaffolds. Attached Figure Description

[0033] 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.

[0034] Figure 1 The images show the fluorescence of hBMSCs at different transfection coefficients. Figure 1 In the diagram, A represents the fluorescence images of hBMSCs at different transfection coefficients. Figure 1 In the image, B represents the cell fluorescence image after 4 days of culture following successful transfection with puromycin.

[0035] Figure 2 The process diagram for preparing the upper layer of the scaffold based on microsphere self-assembly is shown from left to right as follows: microcarrier after adding cell suspension, microspheres dispersed in culture medium, and product after microsphere fusion.

[0036] Figure 3 The results show the effects of TSP1 on HUVEC tube formation and migration. Figure 3 In this context, A represents the result of the endothelial cell tube formation experiment. Figure 3 In this context, B represents the result of the Transwells migration experiment. Figure 3 In this context, C represents the number of tubes formed in the endothelial cell tube formation experiment. Figure 3 In this context, D represents the number of migrating cells as counted in the Transwells migration assay.

[0037] Figure 4 The effect of 5 μg / mL TSP1 on hBMSCs migration, where, Figure 4 A in the image shows the crystal violet staining results of human hBMSCs at the bottom of the Transwell chamber 16 h after migration. The scale bar is 200 μm. Figure 4 In this context, B represents the number of cells counted 16 hours after migration.

[0038] Figure 5 This section describes the preparation process of the printing ink and the morphology of the 3D printed scaffold. Figure 5 In this context, A represents the manufacturing process of COL1 / nHAP ink. Figure 5 B in the image shows the top, bottom, and side views of the lower porous scaffold printed by the 3D bioprinter.

[0039] Figure 6 Experiments were conducted to determine the crosslinking conditions for genipin, in which... Figure 6 Image A shows physical images of ossified scaffolds prepared with different cross-linking times and genipin concentrations, as well as physical images of ossified scaffolds after 2 weeks of simulated in vitro degradation. Figure 6 In this context, B represents the growth state of GFP+hBMSCs in the scaffold.

[0040] Figure 7 The images show the surface morphology of the developmental growth plate layered scaffold. From left to right, they represent the top, bottom, and side surfaces of the developmental growth plate layered scaffold, where U represents the upper layer, M represents the middle layer, and L represents the lower layer.

[0041] Figure 8 The microstructure of the layered scaffold of the developmental growth plate. Figure 8 Image A is an overall electron microscope image of the developmental growth plate layered scaffold, with a scale bar of 500 μm. Figure 8 Image B is a cross-sectional electron microscope image of the developmental growth plate layered scaffold. From left to right, the images are the upper electron microscope image, the middle electron microscope image, and the lower electron microscope image.

[0042] Figure 9 The results of the determination of the mechanical properties of the layered scaffold of the developmental growth plate are as follows. Figure 9 In section A, the statistical results of the reduced modulus of the upper, middle, and lower layers of material are presented. Figure 9 In the middle, B is the modulus fitting diagram of the three-layer material;

[0043] Figure 10 This is a fluorescence image of GFP+hBMSCs after 18 days of culture on a developmental growth plate stratified scaffold. Figure 10 In the diagram, A represents the fluorescence image viewed from the side. Figure 10 In the image, B is the fluorescence image of the three-layer scaffold of the developmental growth plate after artificial peeling. From left to right, the fluorescence images are of the upper scaffold, the middle scaffold, and the lower scaffold. The scale bar is 100 μm.

[0044] Figure 11 The results show the migration of GFP+hBMSCs in a developmental growth plate stratified scaffold;

[0045] Figure 12 The effect of TSP1 on the expression of specific markers SOX8, SOX9 and CD29 in the cell layer was investigated. Figure 12 A in the text is TSP1 - and TSP1 +Immunofluorescence staining results of SOX8 and SOX9 in the upper layer of the developmental growth plate scaffold after 14 days of in vitro chondrogenic induction culture and fixation sectioning, with a scale bar of 50 μm. Figure 12 In the figure, B represents the statistical result of the positive cell rate of SOX8 (ns P > 0.05). Figure 12 C in the figure represents the statistical result of the SOX9 positive cell rate (ns P > 0.05). Figure 12 In the image, D represents CD29 immunofluorescence staining of cells in the upper layer of the developmental growth plate scaffold, with a scale bar of 50 μm.

[0046] Figure 13 This is a model of tibial medial growth plate injury in young rabbits, in which... Figure 13 In the diagram, A represents the construction of a model of tibial medial growth plate injury in young rabbits. Figure 13 B in the diagram represents the surgical implantation of a layered growth plate scaffold.

[0047] Figure 1 4 shows the results of tibial vessel perfusion on the injured side, among which, Figure 14 In the diagram, A represents the tibial vessel perfusion result on the injured side, red represents the reconstructed vessels, and the green area represents the approximate extent of the injury. Figure 14 In the image, B represents the sagittal section of the tibia 3 weeks post-surgery, the red box indicates the damaged area, and microfi1 is shown in blue.

[0048] Figure 15 The above are statistical results of the appearance and deformity of tibial samples 8 weeks post-surgery. Figure 15 Image A shows gross photographs of tibial samples from the injured limb (right) and the opposing limb (left) 8 weeks post-surgery. The scale bar is 2 cm. Figure 15 In the figure, B represents the statistical result of the angular distortion angle. Figure 15 In the figure, C represents the statistical result of tibial shortening. Figure 15 In the diagram, D represents the line connecting the angles of tibial deformity of the injured limb (right) and the opposing limb (left). Figure 15 E in the figure represents the line connecting the lengths of the injured limb (right) and the contralateral limb (left), (ns P>0.05; *P<0.05; **P<0.01);

[0049] Figure 16 The images show the HE staining results 8 weeks post-surgery. The black box indicates the lesion site, and the second row of images shows magnified views of the lesion site. The scale bars are 5000 μm and 500 μm, respectively.

[0050] Figure 17 This image shows a reconstruction of the bone bridge at the injury site and the results of MicroCT analysis. Figure 17In the diagram, A represents the bone bridge reconstruction at the injury site. Using CTan reconstruction analysis software, the surgical site was selected as the area of ​​interest, marked in red, and cropped at the same angle. Only the area of ​​interest was retained, which represents the bone bridge of the damaged growth plate. Figure 1 In Figure 7, B is a statistical chart of bone bridge volume fraction. Figure 17 C in the figure represents the statistical plot of the trabecular bone pattern factor (ns P>0.05; *P<0.05; **P<0.01). Detailed Implementation

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] This invention provides a developmental growth plate layered scaffold comprising upper, middle and lower layers; the upper layer is a chondrocyte layer, the middle layer is a vascular barrier layer, and the lower layer is an ossification layer;

[0057] The chondrocyte layer is loaded with bone marrow mesenchymal stem cells;

[0058] The vascular barrier layer is loaded with platelet-reactive protein 1;

[0059] The ossified layer is composed of type I collagen and hydroxyapatite.

[0060] In this invention, the method for preparing the chondrocyte layer includes the following steps: digesting and resuspending bone marrow mesenchymal stem cells with a cell confluence of 80-90% to obtain a cell resuspension; adding the cell resuspension dropwise onto a microcarrier, allowing it to stand, adding induction differentiation medium, dispersing the cells, replacing the induction differentiation medium, and incubating to obtain the chondrocyte layer. The density of the cell resuspension is preferably 1-3 × 10⁻⁶. 6 Cells / mL, more preferably 2×10 6 The concentration of the culture medium is 100 cells / mL; the preferred temperature for standing is 37°C; the preferred standing time is 2-3 hours, more preferably 2.5 hours; the preferred frequency for changing the induction differentiation medium is 2-3 times per day, more preferably 2.5 times per day; the preferred incubation time is 5-7 days, more preferably 6 days.

[0061] In this invention, hBMSCs secrete numerous matrix proteins under static chondrogenic induction conditions. As the matrix proteins increase, the microspheres in the culture medium gradually fuse and assemble into a cohesive whole. On day 7 of induction, the microsphere aggregates become more tightly packed, translucent, and elastic, resembling the appearance of natural hyaline cartilage (e.g., hBMSCs). Figure 2 (As shown).

[0062] In this invention, the method for preparing the vascular barrier layer includes the following steps: dissolving methacryloyl gelatin and a photoinitiator separately, adding platelet-reactive protein 1, and irradiating with blue light or ultraviolet light to obtain the vascular barrier layer. Preferably, a phosphate-buffered saline solution is used for dissolution, and the photoinitiator is preferably lithium phenyl-2,4,6-trimethylbenzoylphosphine. The photoinitiator is preferably dissolved in PBS to obtain a photoinitiator solution with a mass-volume concentration of 0.2–0.3%. The mass-volume concentration of the photoinitiator is further preferably 0.25%. After dissolution, the solution is preferably heated at 60–70°C for 20–30 min, with the heating temperature preferably being 64–68°C, more preferably 65°C. The heating time is preferably 24–28 min, more preferably 25 min. After heating and dissolution, the solution is preferably placed in a syringe and filtered into an EP tube. The pore size of the filter is 0.22 μm. The effective concentration of platelet-reactive protein 1 is preferably 5 μg / mL.

[0063] In this invention, the method for preparing the ossification layer includes the following steps: dissolving type I collagen, adding hydroxyapatite to obtain 3D printing bio-ink, adding it to a 3D bioprinter for printing to obtain a type I collagen / hydroxyapatite scaffold; freeze-drying the type I collagen / hydroxyapatite scaffold, cross-linking it, and obtaining the ossification layer. Acetic acid is preferably used for dissolution; the concentration of acetic acid is preferably 0.04-0.06M, more preferably 0.05M; the printing size is preferably 5×5×2mm, the line spacing is preferably 0.5-0.8mm, more preferably 0.6mm; the printing speed is preferably 6-10mm / s, more preferably 8mm / s; the mass ratio of type I collagen to hydroxyapatite is preferably 2:1; the freeze-drying time is preferably 4-6h, more preferably 5h; genipin is preferably used for crosslinking; the mass-volume ratio of genipin is preferably 0.1%; the crosslinking time is preferably 20h; after crosslinking, ethanol and saturated glycine solution are preferably used for washing in sequence; the number of washings is preferably 2-4 times, more preferably 3 times; the volume fraction of ethanol is preferably 70-80%, more preferably 74-78%, even more preferably 75%; the washing time is preferably 1.5-2.5h, more preferably 2h.

[0064] In this invention, type I collagen sponge is cut into smaller pieces to increase the contact area with the acetic acid solution, facilitating its complete dissolution and preventing it from clumping together. The completely dissolved collagen gel is translucent. After adding nHAP, it becomes milky white and has strong viscosity. Using the prepared COL I / nHAP ink for 3D printing, the printed scaffold is as follows... Figure 5 As shown in Figure B, the printed support has dimensions of 5×5×2mm (length×width×height), with a regularly arranged mesh structure. The ink can be smoothly extruded and printed, resulting in a smooth and stable support structure. The pores on the top and bottom surfaces of the support are of uniform size, and the side structure is clear, indicating that the COL I / nHAP (2:1) ink configured in this invention has good printability, and the lower layer structure of the layered support prepared thereby is relatively uniform.

[0065] The present invention also provides a method for assembling the aforementioned developmental growth plate layered scaffold, comprising the following steps:

[0066] The chondrocyte layer was cut into 5×5mm pieces and adhered to the vascular barrier layer to obtain a pre-assembled scaffold. A polyvinylidene fluoride membrane was inserted from one side of the pre-assembled scaffold, and the insertion was stopped at 1 / 2 of the pre-assembled scaffold. The pre-assembled scaffold was then lifted and pushed onto the ossified layer for cross-linking. It was then transferred to a chondrogenic induction culture medium for culture to obtain the developmental growth plate layered scaffold.

[0067] In this invention, the preparation of the developmental growth plate layered scaffold is preferably carried out on a 3D bioprinter under negative pressure; the cross-linking is preferably carried out under a 405nm light source; the cross-linking time is preferably 14-16s, more preferably 15s; the culture temperature is preferably 37℃; and the culture time is preferably 5-7d, more preferably 6d.

[0068] The present invention also provides the application of the aforementioned developmental growth plate layered scaffold in the preparation of products for repairing epiphyseal injuries.

[0069] Example 1

[0070] A developmental growth plate layered scaffold consists of three layers: upper, middle, and lower; the upper layer is a chondrocyte layer, the middle layer is a vascular barrier layer, and the lower layer is an ossification layer.

[0071] The chondrocyte layer is loaded with bone marrow mesenchymal stem cells;

[0072] The vascular barrier layer is loaded with platelet-reactive protein 1;

[0073] The ossified layer is composed of type I collagen and hydroxyapatite;

[0074] The chondrocyte layer was prepared as follows: hBMSCs (purchased from OriCel1) were cultured in an incubator at 37°C with 5% CO2. When the cell confluence reached 80%, the culture medium was aspirated and the cells were washed twice with PBS solution. 1 mL of 0.05% trypsin was added for digestion and resuspending. The cells were passaged at a ratio of 1:3 and cultured in an incubator at 37°C with 5% CO2 until a sufficient number of hBMSCs were obtained. The cells were then digested, resuspended, and a density of 2 × 10⁶ cells / mL was obtained. 6Cell resuspension was prepared at a density of 90,000 cells / mL. 50 μL of cell resuspension was added to the center of a low-adhesion six-well plate. A 20 mg microcarrier (approximately 90,000 gelatin microspheres, purchased from Beijing Huakan Biotechnology Co., Ltd.) was placed on the cell resuspension. 300 μL of the well-mixed cell resuspension was then slowly added dropwise at a rate of 300 μL / well to the microcarrier. The remaining liquid was added evenly from the periphery of the microcarrier to ensure it was fully saturated without disintegrating. 2 mL of PBS solution was then added to the wells of the six-well plate to minimize cell resuspension loss. The plate was then incubated at 37°C for 3 hours to allow complete adhesion of hBMSCs to the gelatin microspheres, thus obtaining the hBMSCs. MSCs microspheres; hBMSCs chondrogenic differentiation induction medium (purchased from OriCell, China) was prepared under light-protected conditions. A six-well plate was removed and hBMSCs chondrogenic differentiation induction medium was added at a rate of 5 mL / well in a biosafety cabinet. The hBMSCs microspheres were gently dispersed using a 200 μL pipette tip to ensure complete separation and uniform distribution within the culture plate without obvious clumps. The hBMSCs chondrogenic differentiation induction medium was then replaced every 2 days for a total of 7 days. The dispersed microcarriers gradually fused and assembled into a translucent whole, yielding the chondrocyte layer, which serves as the upper layer of the composite scaffold.

[0075] The method for preparing the vascular barrier layer is as follows: 0.5g of shredded methacryloyl gelatin (GelMA) dry matter and 0.2% (w / v) phenyl-2,4,6-trimethylbenzoyl lithium phosphine were dissolved in PBS buffer. The solution was heated in a 60°C water bath for 30 minutes, during which time the solution could be removed and shaken to accelerate dissolution. After dissolution, the solution was immediately transferred to a clean bench. A 0.22μm filter was attached to a 5mL syringe, the stopcock was removed, GelMA solution was poured in, the stopcock was replaced, and the GelMA solution was filtered into a sterile EP tube. The solution was stored at -20°C protected from light. The filtration process should be as rapid as possible to prevent low-temperature gelation and clogging of the needle. 5μg / mL of platelet-reactive protein 1 (TSP1) was added, and the solution was placed in a 37°C water bath to convert it into a sol. The printing specifications were set to 5mm × 5mm × 0.5mm, with 2 layers, a nozzle temperature of 25°C, and a platform temperature of room temperature. The vascular barrier layer was obtained by 3D printing.

[0076] The preparation method of the ossified layer is as follows: 2g of type I collagen (COL I) from pigskin is cut into small pieces and dissolved in 0.05M acetic acid. The solution can be placed in a 4℃ refrigerator to aid dissolution until a semi-transparent gel is formed. 1g of hydroxyapatite (nHAP) is added in small amounts several times while stirring to ensure uniform dispersion of nHAP in the collagen gel, thus obtaining 3D printing bio-ink. This bio-ink is then added to the printing syringe of a 3D bioprinter, fitted with a 25G needle, and the printing dimensions are set to 5×5×2mm, line spacing to 0.6mm, and a speed to 8mm / s. After calibrating the platform height, printing can begin. The printed type I collagen / hydroxyapatite... The apatite scaffold was stored at -80°C. The type I collagen / hydroxyapatite scaffold was freeze-dried for 4 hours, then immersed in 0.1% genipin at 20°C for 20 hours for crosslinking. After removing the scaffold, it was immersed in 75% ethanol and washed on a shaker for 2 hours, followed by washing with saturated glycine solution on a shaker for 2 hours. This washing was repeated three times, followed by washing with pure water three times to obtain the ossified layer. The ossified layer was then freeze-dried and stored. Before assembly, the ossified layer was immersed in 75% ethanol overnight. The ossified layer was then removed and washed three times with sterile PBS.

[0077] On a 3D bioprinter under negative pressure, the chondrocyte layer was removed, the surface culture medium was carefully absorbed with gauze, and the cells were cut into 5×5mm pieces with a sterile blade. The cells were then carefully picked up with forceps and gently adhered to the vascular barrier layer to obtain a pre-assembled scaffold. A PVDF membrane was inserted from one side of the pre-assembled scaffold, and the insertion was stopped at about 1 / 2 of the pre-assembled scaffold and carefully lifted. With the assistance of flat-tipped forceps, the membrane was gently pushed onto the ossification layer and gently pressed to ensure complete adhesion. The membrane was placed under a 405nm light source for cross-linking for 15s and transferred to a 24-well plate containing human BMSC chondrogenic induction medium (purchased from Cyagen Biosciences). The 24-well plate was then transferred to a clean bench, the medium was discarded, and 1mL of PBS containing 2% PSA was added. The plate was washed twice, the PBS was discarded, and 1mL of fresh human BMSC chondrogenic induction medium was added to each well for induction culture. The human BMSC chondrogenic induction medium was replaced every 2 days and cultured at 37℃ for 7 days to obtain the developmental growth plate layered scaffold.

[0078] Example 2

[0079] A developmental growth plate layered scaffold consists of three layers: upper, middle, and lower; the upper layer is a chondrocyte layer, the middle layer is a vascular barrier layer, and the lower layer is an ossification layer.

[0080] The chondrocyte layer is loaded with bone marrow mesenchymal stem cells;

[0081] The vascular barrier layer is loaded with platelet-reactive protein 1;

[0082] The ossified layer is composed of type I collagen and hydroxyapatite.

[0083] The chondrocyte layer was prepared as follows: hBMSCs (purchased from OriCell) were cultured in an incubator at 37°C with 5% CO2. When the cell confluence reached 90%, the culture medium was aspirated, and the cells were washed twice with PBS solution. 1 mL of 0.05% trypsin was added for digestion and resuspending. The cells were passaged at a 1:2 ratio and cultured in an incubator at 37°C with 5% CO2 until a sufficient number of hBMSCs were obtained. The cells were then digested, resuspended, and a density of 1×10⁶ cells / mL was obtained. 6 Cell resuspension was prepared at a density of 90,000 cells / mL. 50 μL of cell resuspension was added to the center of a low-adhesion six-well plate. A 20 mg microcarrier (approximately 90,000 gelatin microspheres, purchased from Beijing Huakan Biotechnology Co., Ltd.) was placed on the cell resuspension. 300 μL of the well-mixed cell resuspension was then slowly added dropwise at a rate of 300 μL / well to the microcarrier. The remaining liquid was added evenly from the periphery of the microcarrier to ensure it was fully saturated without disintegrating. 2 mL of PBS solution was then added to the wells of the six-well plate to minimize cell resuspension loss. The plate was then incubated at 37°C for 2 hours to allow complete adhesion between the hBMSCs and the gelatin microspheres, thus obtaining the hBMSCs. MSCs microspheres; hBMSCs chondrogenic differentiation induction medium (purchased from OriCell, China) was prepared under light-protected conditions. A six-well plate was removed and hBMSCs chondrogenic differentiation induction medium was added at a rate of 5 mL / well in a biosafety cabinet. The hBMSCs microspheres were gently dispersed using a 200 μL pipette tip to ensure complete separation and uniform distribution within the culture plate without obvious clumps. The hBMSCs chondrogenic differentiation induction medium was then replaced every 1 day for a total of 7 days. The dispersed microcarriers gradually fused and assembled into a translucent whole, yielding the chondrocyte layer, which serves as the upper layer of the composite scaffold.

[0084] The method for preparing the vascular barrier layer is as follows: 0.5g of shredded methacryloyl gelatin (GelMA) dry matter and 0.2% (w / v) phenyl-2,4,6-trimethylbenzoyl lithium phosphine were dissolved in PBS buffer and heated in a 70°C water bath for 20 minutes. During this time, the solution could be removed and shaken to accelerate dissolution. After dissolution, the solution was immediately transferred to a clean bench. A 0.22μm filter was attached to a 5mL syringe, the stopcock was removed, GelMA solution was poured in, the stopcock was replaced, and the GelMA solution was filtered into a sterile EP tube and stored at -20°C protected from light. The filtration process should be as rapid as possible to prevent low-temperature gelation and needle clogging. 10μg / mL of platelet-reactive protein 1 (TSP1) was added and placed in a 37°C water bath to convert it into a sol. The printing specifications were set to 5mm × 5mm × 0.5mm, with 2 printing layers, a nozzle temperature of 25°C, and a platform temperature of room temperature. The vascular barrier layer was obtained through 3D printing.

[0085] The preparation method of the ossified layer is as follows: 2g of type I collagen (COL I) from pigskin is cut into small pieces and dissolved in 0.04M acetic acid. The solution can be placed in a 4℃ refrigerator to aid dissolution until a semi-transparent gel is formed. 1g of hydroxyapatite (nHAP) is added in small amounts several times while stirring to ensure uniform dispersion of nHAP in the collagen gel, thus obtaining 3D printing bio-ink. This bio-ink is then added to the printing syringe of a 3D bioprinter, fitted with a 25G needle, and the printing dimensions are set to 5×5×2mm, line spacing to 0.5mm, and a speed to 6mm / s. After calibrating the platform height, printing can begin. The printed type I collagen / hydroxyapatite... The ossified scaffold was stored at -80°C. The type I collagen / hydroxyapatite scaffold was freeze-dried for 5 hours, then immersed in 0.1% genipin at 25°C for 20 hours for crosslinking. After removing the scaffold, it was immersed in 70% ethanol and washed on a shaker for 2 hours. Then it was washed with saturated glycine solution on a shaker for 1.5 hours, for a total of 2 washes. Finally, it was washed three times with pure water to obtain the ossified layer, which was then freeze-dried and stored. Before assembly, the ossified layer was immersed in 75% ethanol overnight. The ossified layer was then removed and washed three times with sterile PBS.

[0086] On a 3D bioprinter under negative pressure, the chondrocyte layer was removed, the surface culture medium was carefully absorbed with gauze, and the cells were cut into 5×5mm pieces with a sterile blade. The cells were then carefully picked up with forceps and gently adhered to the vascular barrier layer to obtain a pre-assembled scaffold. A PVDF membrane was inserted from one side of the pre-assembled scaffold, stopping at approximately half its length and carefully lifted. With the assistance of flat-tipped forceps, the membrane was gently pushed onto the ossification layer, gently pressed to ensure complete adhesion, and cross-linked under a 405nm light source for 14s. The membrane was then transferred to a 24-well plate containing human BMSC chondrogenic induction medium (purchased from Cyagen). The 24-well plate was then transferred to a clean bench, the medium was discarded, and 1 mL of PBS containing 2% PSA was added. The plate was washed twice, the PBS was discarded, and 1 mL of fresh human BMSC chondrogenic induction medium was added to each well for induction culture. The human BMSC chondrogenic induction medium was replaced every 2.5 days, and the plate was cultured at 37°C for 6 days to obtain the developmental growth plate layered scaffold.

[0087] Example 3

[0088] A developmental growth plate layered scaffold consists of three layers: upper, middle, and lower; the upper layer is a chondrocyte layer, the middle layer is a vascular barrier layer, and the lower layer is an ossification layer.

[0089] The chondrocyte layer is loaded with bone marrow mesenchymal stem cells;

[0090] The vascular barrier layer is loaded with platelet-reactive protein 1;

[0091] The ossified layer is composed of type I collagen and hydroxyapatite;

[0092] The chondrocyte layer was prepared as follows: hBMSCs (purchased from OriCell) were cultured in an incubator at 37°C with 5% CO2. When the cell confluence reached 85%, the culture medium was aspirated and the cells were washed twice with PBS solution. 1 mL of 0.05% trypsin was added for digestion and resuspending. The cells were passaged at a ratio of 1:4 and cultured in an incubator at 37°C with 5% CO2 until a sufficient number of hBMSCs were obtained. The cells were then digested. The cells were resuspended to obtain a cell resuspension with a density of 3 × 10⁶ cells / mL. 50 μL of the cell suspension was added dropwise to the center of a low-adhesion six-well plate. A 20 mg microcarrier (approximately 90,000 gelatin microspheres, purchased from Beijing Huakan Biotechnology Co., Ltd.) was placed on the cell suspension. 300 μL of the well-mixed cell resuspension was then slowly added dropwise at a rate of 300 μL / well to the microcarrier. The remaining liquid was added evenly from the periphery of the microcarrier to ensure it was fully saturated without disintegrating. Finally, 2 mL of the microcarrier was added to each well of the six-well plate. PBS solution was used to reduce the loss of cell resuspension in the microcarriers. The microcarriers were then incubated at 37°C for 2 hours to allow complete adhesion between hBMSCs and gelatin microspheres, resulting in hBMSC microspheres. hBMSC chondrogenic differentiation induction medium (purchased from OriCell, China) was prepared under light-protected conditions. The medium was added to each well of a six-well plate at a rate of 5 mL / well in a biosafety cabinet. The hBMSC microspheres were gently dispersed using a 200 μL pipette tip to ensure complete separation and uniform distribution within the plate, without any obvious clumps. The medium was then replaced every 1.5 days for a total of 7 days. The dispersed microcarriers gradually fused and assembled into a translucent whole, yielding the chondrocyte layer, which serves as the upper layer of the composite scaffold.

[0093] The method for preparing the vascular barrier layer is as follows: 0.5g of shredded methacryloyl gelatin (GelMA) dry matter and 0.2-0.3% (w / v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate are dissolved in PBS buffer and heated in a 65°C water bath for 25 minutes. During this time, the solution can be removed and shaken to accelerate dissolution. After dissolution, the solution is immediately transferred to a clean bench. A 0.22μm filter is attached to a 5mL syringe, the stopcock is removed, GeMA solution is poured in, the stopcock is replaced, and the GelMA solution is filtered into a sterile EP tube and stored at -20°C protected from light. The filtration process should be as rapid as possible to prevent low-temperature gelation and needle clogging. 8μg / mL of platelet-reactive protein 1 (TSP1) is added and placed in a 37°C water bath to convert it into a sol. The printing specifications are set to 5mm × 5mm × 0.5mm, with 2 printing layers, a nozzle temperature of 25°C, and a platform temperature of room temperature. The vascular barrier is obtained through 3D printing.

[0094] The preparation method of the ossified layer is as follows: 2g of type I collagen (COL I) from pigskin is cut into small pieces and dissolved in 0.06M acetic acid. The solution can be placed in a 4℃ refrigerator to aid dissolution until a semi-transparent gel is formed. 1g of hydroxyapatite (nHAP) is added in small amounts several times while stirring to ensure uniform dispersion of nHAP in the collagen gel, thus obtaining 3D printing bio-ink. This bio-ink is then added to the printing syringe of a 3D bioprinter, fitted with a 25G needle, and the printing dimensions are set to 5×5×2mm, line spacing to 0.8mm, and a speed to 10mm / s. After calibrating the platform height, printing can begin. The printed type I collagen / hydroxyapatite... The apatite scaffold was stored at -80°C. The type I collagen / hydroxyapatite scaffold was freeze-dried for 6 hours, then immersed in 0.1% genipin at 30°C for 20 hours for crosslinking. After removing the scaffold, it was immersed in 80% ethanol and washed on a shaker for 2 hours. Then it was washed with saturated glycine solution on a shaker for 2.5 hours, for a total of 4 washes. Finally, it was washed three times with pure water to obtain the ossified layer, which was then freeze-dried and stored. Before assembly, the ossified layer was immersed in 75% ethanol overnight. The ossified layer was then removed and washed three times with sterile PBS.

[0095] On a 3D bioprinter under negative pressure, the chondrocyte layer was removed, the surface culture medium was carefully absorbed with gauze, and the cells were cut into 5×5mm pieces with a sterile blade. The cells were then carefully picked up with forceps and gently adhered to the vascular barrier layer to obtain a pre-assembled scaffold. A PVDF membrane was inserted from one side of the pre-assembled scaffold, stopping at approximately half its length and carefully lifted. With the assistance of flat-tipped forceps, the membrane was gently pushed onto the ossification layer, and gently pressed to ensure complete adhesion. The scaffold was cross-linked for 16 seconds under a 405nm light source and transferred to a 24-well plate containing human BMSC chondrogenic induction medium (purchased from Cyagen Biosciences). The 24-well plate was then transferred to a clean bench, the medium was discarded, and 1 mL of PBS containing 2% PSA was added. The plate was washed twice, the PBS was discarded, and 1 mL of fresh human BMSC chondrogenic induction medium was added to each well for induction culture. The human BMSC chondrogenic induction medium was replaced every 3 days, and the plate was cultured at 37°C for 5 days to obtain the developmental growth plate layered scaffold.

[0096] Experiment 1: Optimization of Transfection Coefficient

[0097] To track the migration and differentiation behavior of hBMSCs, this invention labeled hBMSCs with green fluorescent protein (GFP) via lentiviral transfection and investigated the effects of MOIs of 10, 30, 50, and 100 on transfection efficiency and cell proliferation. Simultaneously, successfully labeled hBMSCs were screened using puromycin and seeded at the same density. Fluorescence images were taken on day 1 and day 4 post-seeding to observe cell morphology and density. Results are as follows: Figure 1 As shown.

[0098] The results showed that the green fluorescence expression in the MOI-10 group was poor, and the transfection efficiency was too low; therefore, the MOI-10 group was excluded. Compared with the MOI-30 group, the cell density in the MOI-50 and MOI-100 groups was lower, and the cell morphology in the MOI-100 group was significantly altered. Considering both transfection efficiency and cell state, MOI-30 was the optimal transfection coefficient for hBMSCs.

[0099] Experimental Example 2: Selection of Effective Concentration of TSP1

[0100] 2.1 Endothelial cell tube formation experiment

[0101] Prepare 96-well cell culture plates and sterile 200 μL pipette tips, and pre-cool them overnight at 4°C. At the same time, place Matrigel (purchased from Coming / g / BD, catalog number 356234) in a 4°C refrigerator overnight to fully dissolve it.

[0102] Lay Matrigel on the plate. Prepare ice packs and place them on the clean bench. Take out the pre-chilled pipette tips, 96-well culture plates, and Matrigel, and place them on ice. Continue the process on ice. Using the pre-chilled pipette tips, add 50 μL of Matrigel to each well of the 96-well plate, avoiding air intake during pipetting to prevent air bubbles from forming in the Matrigel. Ensure the pipette tip is perpendicular to the culture plate during addition, and try to use the same pipette tip for each experiment. After pipetting, let the 96-well plate stand on the platform for a few minutes, then incubate at 37°C for 0.5 h. Prepare human umbilical cord endothelial cells (HUVEC, purchased from Cyagen) in the logarithmic growth phase (within the fifth generation). Digest the cells with 0.25% EDTA-containing trypsin (purchased from Gibco) until the cells shrink. Dilute the cell concentration to 2 × 10⁻⁶ using serum-free endothelial cell culture medium (purchased from Cyagen). 5 Cells / mL. Add 100 μl of cell suspension to each well and gently shake to distribute cells evenly. Incubate at 37℃, 5% CO2. Tube formation can be observed after 6 hours, with optimal formation at 8 hours. Images of each group were acquired at 8 hours. The number of tube nodes was calculated using ImageJ software. A control group (i.e., 0 μg / mL) was also set up. Results are as follows: Figure 3 A and Figure 3 As shown in C.

[0103] The results showed that the inhibitory effect of TSP1 on tubule formation was concentration-dependent. Compared with the control group (i.e., 0 μg / mL), HUVECs were basically unable to form tubules under the intervention of 5 μg / mL TSP1. The tubular structures and total length were significantly reduced, and the number of fulcrums decreased from (75±10) per field of view to (35±10) per field of view (p<0.05).

[0104] 2.2 Transwell migration experiment

[0105] HUVECs (purchased from Cyagen) were resuspended in serum-free culture medium to achieve a cell concentration of 1.5 × 10⁻⁶ cells. 5 Serum-free cell suspension was obtained by increasing the cell density to 100 μg / mL. The serum-free cell suspension was seeded into the upper chamber of a Transwell microarray, and 500 μl of complete culture medium (purchased from Cyagen) was added to the lower chamber. The microarray was incubated at 37°C with 5% CO2 for 16 h. The microarray was then removed, and the cells were gently rinsed three times with PBS to remove the culture medium. After discarding the PBS, 4% paraformaldehyde was added for fixation at room temperature for 20 min, followed by three washes with PBS. Crystal violet staining was performed by immersing the microarray in crystal violet solution for 15 min, followed by three washes with PBS and air drying. Cells were carefully wiped from the microarray with a moistened cotton swab and observed and photographed under an inverted microscope. Cell counts were calculated using ImageJ software. A control group (0 μg / mL) was also set up. This experiment was repeated with hBMSCs under identical conditions to HUVECs, and the results are shown below. Figure 3 B and Figure 3 D and Figure 4 As shown.

[0106] The results showed that both the 2 μg / mL and 5 μg / mL TSP1 groups had significant inhibitory effects on angiogenesis compared to the control group. In summary, the 5 μg / mL TSP1 group showed the best inhibitory effect on angiogenesis. Furthermore, the morphology of migrating cells at the 5 μg / mL concentration was not significantly different from the control group, both exhibiting the typical long spindle shape of hBMSCs. The number of migrating cells showed no significant difference between the two groups. This demonstrates that 5 μg / mL TSP1 has no effect on hBMSC migration and can be used for the construction of developmental growth plate stratified scaffolds.

[0107] Experiment 3: Optimization of Genipin Concentration and Crosslinking Time

[0108] Collagen peptide chains are linked by hydrogen bonds, which are non-covalent interactions and therefore unstable. Consequently, the printed COL I / nHAP scaffold will gradually collapse in aqueous solutions such as culture media. Therefore, this invention uses genipin, a natural biocrosslinking agent, to stabilize collagen into chemical chains. Compared to chemical crosslinking agents such as glutaraldehyde, genipin offers milder reaction conditions, better biocompatibility, and anti-inflammatory and antibacterial effects. However, excessive genipin can inhibit cell proliferation, thus requiring the exploration of optimal genipin crosslinking conditions.

[0109] This invention sets two concentrations (0.1% and 0.5%) and four crosslinking times (2, 4, 8, and 20 hours) to prepare ossification layer scaffolds according to the method in Example 1. The results are as follows... Figure 6 As shown in A in the diagram.

[0110] The results showed that the structure of the COL I / nHAP scaffold became increasingly stable with increasing genipin concentration and cross-linking time. The scaffolds under cross-linking conditions of (0.1%, 20 h) and (0.5%, 8 h) did not show significant structural changes after in vitro degradation, meeting the requirements for subsequent experiments. Therefore, these two conditions were chosen to test its cytotoxicity.

[0111] Experiment 4: Cell Adhesion and Viability Assay

[0112] Human bone marrow mesenchymal stem cells (hBMSCs) were selected as the research object. To track hBMSCs cells, they were transfected with lentivirus (purchased from Hanheng Biotechnology Co., Ltd.) and labeled with green fluorescence, obtaining a density of 5 × 10⁻⁶ cells. 5 Prepare a GFP+hBMSCs cell suspension of 1 cell / mL for later use.

[0113] Osteoformed layers were prepared according to the method described in Example 1, with the concentration and time of genipin crosslinking set at (0.5%, 8 h) and (0.1%, 20 h), respectively. The sterilized ossified layers were placed in a GFP+hBMSCs cell suspension and observed under a fluorescence microscope. After approximately 6 h, when the cells adhered to the scaffold, the scaffold was removed and placed in a 24-well plate containing fresh culture medium (human BMSCs medium, purchased from Cyagen). After 20 h, images were taken using an inverted fluorescence microscope. The results are as follows: Figure 6 As shown in B in the diagram.

[0114] The results showed that the scaffold exhibited significant toxicity to cells under the (0.5%, 8h) crosslinking condition, causing cell shrinkage and marked morphological changes. In contrast, under the (0.1%, 20h) crosslinking condition, the pseudopodia of cells in the scaffold extended well, and cells grew along the printing column. In conclusion, the COL I / nHAP scaffold under the (0.1%, 20h) crosslinking condition demonstrates certain anti-degradation capabilities and good biocompatibility, meeting the requirements for subsequent cell culture and in vivo repair experiments.

[0115] Comprehensive characterization of developmental growth plate layered scaffolds in Experiment Example 5

[0116] 5.1 Measure the composite three-layer support using vernier calipers. The measurement results are as follows: Figure 7 As shown.

[0117] The results showed that the upper and middle layers of the composite three-layer scaffold were both 0.5 mm thick, while the lower layer was 1 mm thick. Furthermore, the 3D-printed lower scaffold exhibited large, visible pores, supporting the permeation of nutrients.

[0118] 5.2 Scanning electron microscopy for microstructure analysis

[0119] The developmental growth plate scaffold samples prepared in Example 1 and cultured for 14 days were washed three times with pure water to remove surface impurities. They were then transferred to 12-well plates and fixed with 4% paraformaldehyde for 2 hours. After fixation, they were washed three times with PBS solution for 5 minutes each time. Residual moisture was absorbed, and the plates were frozen at -80°C for 2 hours. The 12-well plates were then opened and freeze-dried for 12 hours. The plates were then closed and sealed with one or two turns of sealing film to prevent moisture from affecting the scanning results. The samples were then placed in the gold sputtering chamber for gold sputtering. The samples were placed in the sample chamber according to the markings, adjusted to the correct position, and observed and photographed at 200x and 10000x magnification. The results are as follows: Figure 8 As shown.

[0120] The results showed that the three layers of material were tightly connected and had different pore characteristics. The upper layer had uniform and interconnected pores, the middle layer had larger pores, and the lower layer was a 3D-printed structure with a controllable macroporous structure and a rough pore surface suitable for cell adhesion. The pore size of the three-layer scaffold gradually increased from the upper to the lower layers, with the middle layer acting as a bridge connecting the upper and lower layers. The microstructure of the upper and lower layers was conducive to cell adhesion and growth.

[0121] 5.3 Determination of Mechanical Properties

[0122] The mechanical properties of the scaffold were measured using a bio-nanoindentation instrument (Optic11BV). Statistical results are as follows: Figure 9 As shown.

[0123] The results showed that the mechanical properties of the three layers also differed. The reduced moduli of the upper and middle layers were similar, approximately 25 kPa, while the modulus of the lower layer was approximately 700 kPa. The mechanical properties of the lower layer were close to those of the natural growth plate (data from J Mech Behav Biomed Mater. 2022. 128: 105-102). The three-dimensional fitting plot of the composite scaffold's modulus showed a significant gradient difference, with the elastic modulus gradually increasing from the upper to the lower layers.

[0124] 5.4 Determination of cell distribution and migration

[0125] The GFP+hBMSCs prepared in Experiment 4 were seeded on microcarriers (gelatin microspheres) and incubated in proliferation medium (purchased from Cyagen) for 18 days. The distribution of green fluorescent cells was then observed from the side of the developmental growth plate stratified scaffold.

[0126] like Figure 10 As shown, the cells are arranged longitudinally. Subsequently, the upper, middle, and lower layers of the composite scaffold were artificially separated. From a top-down view, it was observed that the upper layer cells grew along the microspheres with a high cell density, the middle layer cells were evenly distributed, and the lower layer scaffold edges showed green fluorescent cells. These results indicate that, when incubated in proliferation medium, cells in the upper layer of the stratified scaffold can migrate to the lower layer and colonize.

[0127] 5.5 Cell migration under chondrogenic induction culture conditions

[0128] To further investigate whether hBMSCs could still migrate in chondrogenic induction medium using a developmental growth plate layered scaffold, lentivirus was used to transfect green fluorescently labeled hBMSCs to obtain GFP+-hBMSCs. These GFP+-hBMSCs were then seeded onto microcarriers to obtain green fluorescent microcarriers (i.e., cell microspheres). The middle and lower layers of the developmental growth plate layered scaffold were then prepared and assembled according to the method described in Example 1. The developmental growth plate layered scaffold was cultured in chondrogenic differentiation induction medium (purchased from Cyagen) for 14 days, and the cell nuclei were stained after sectioning. The results are as follows: Figure 11 As shown.

[0129] At 8 days of induction culture, only a small number of positive nuclear staining samples were observed at the edge of the lower layer adjacent to the anti-angiogenic layer. After 14 days of chondrogenic induction culture, the number of positive nuclear staining samples in the lower layer increased significantly and the distribution became more uniform. In summary, this demonstrates that under both proliferation and chondrogenic induction conditions, GFP+hBMSCs can penetrate the anti-angiogenic layer (middle layer) and colonize in the lower layer. However, it is regrettable that positive nuclear staining was only observed in the middle layer of some sections. This is presumably because the middle layer has insufficient dry matter, limited space for cell growth, and the limited section thickness makes it difficult to observe cells.

[0130] 5.6 Immunofluorescence staining (SOX8, SOX9, CD29)

[0131] SOX8 and SOX9 are both key transcription factors for chondrocyte differentiation. They are expressed in the cell nucleus and indicate the differentiation of hBMSCs into chondrocytes. CD29 is a specific marker for hBMSCs, representing undifferentiated hBMSCs.

[0132] A developmental growth plate layered scaffold unloaded with TSP1 was prepared according to the method in Example 1, and denoted as TSP1. - The developmental growth plate layered scaffold prepared in Example 1 was used as TSP1. + .

[0133] Frozen sections from the developmental growth plate scaffold were brought to room temperature and washed three times with PBS for 5 min each time. The tissue was circled with a histochemical pen, and covered with immunofluorescence strong permeabilization buffer (purchased from Beyotime Biotechnology). Permeabilization was performed at room temperature for 10 min, followed by three washes with PBS for 5 min each time. Immunofluorescence staining blocking buffer (purchased from Beyotime Biotechnology) was used for blocking at 37°C for 30 min, followed by one wash with PBS. Antibody dilution buffer (SOX8, SOX9, CD29, antibodies purchased from Abcam) was prepared at a 1:100 ratio, added to the tissue, and incubated overnight at 4°C, followed by three washes with PBS for 5 min each time. Secondary antibody dilution buffer (purchased from Beyotime Biotechnology) was prepared at a 1:200 ratio, added to the tissue, and incubated at 37°C for 1 h. The tissue was washed three times with PBS for 10 min each time. The slides were mounted with DAPI anti-fluorescence quenching mounting buffer (purchased from Beyotime Biotechnology), observed under a fluorescence microscope, and photographed. Results are as follows: Figure 12 As shown.

[0134] The results showed that TSP1 - Group and TSP1 + The expression levels of SOX8 and SOX9 in the cell layers were similar. CD29 staining results indicated the presence of some undifferentiated hBMSCs in the upper scaffold layer, i.e., the chondrocyte layer. This coexistence of differentiated chondrocytes and undifferentiated stem cells is similar to the cell state in the natural growth plate.

[0135] Experimental Example 6: In vivo application of a developmental growth plate layered scaffold

[0136] 6.1 Construction of the growth plate defect model

[0137] Developmental growth plate layered scaffolds without TSP1 loading were prepared as a comparative example according to the method in Example 1. The developmental growth plate layered scaffolds prepared in Example 1 were cultured in chondrogenic induction medium (purchased from Cyagen). On the day of surgery, the two groups of layered scaffolds (TSP1- group and TSP+ group) were removed from the incubator and placed in a 4°C refrigerator for later use. The experiment consisted of three groups: TSP1... + Scaffold assembly (the developmental growth plate layered scaffold prepared in Example 1), TSP1 - The scaffold group (comparative example) and the blank control group (Blank group, no material was implanted at the defect site).

[0138] Modeling diagram and operation process as follows Figure 13As shown. Six-week-old male New Zealand White rabbits (purchased from Air Force Medical University), weighing approximately 1.5 kg, were selected as the research subjects. The rabbits were anesthetized by injecting 3% sodium pentobarbital into the marginal ear vein, with an anesthetic dosage of 1 mL / kg. After confirming the absence of corneal reflex, the skin was prepared by shaving the hair within a 5 cm radius around the surgical site on the medial side of the right knee joint to expose the skin. The prepared area was disinfected with iodine-soaked cotton balls, three times from the inside out, and a drape was applied. Lidocaine (provided by the Animal Center of Air Force Medical University) was injected subcutaneously into the surgical area to create a 2 cm × 1 cm wheal, which was massaged until absorbed. 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-shaped white tendon on the medial side of the knee joint was freed and fixed with a retractor, fully exposing the medial growth plate of the tibia in the rabbit's right hind limb. The growth plate was disrupted using a pointed scalpel, creating a lesion strip approximately 3 mm high, 5 mm wide, and 5 mm deep. A layered growth plate scaffold was inserted into the lesion strip (the upper layer near the proximal tibia, and the lower layer near the metaphysis). The muscle was sutured to completely cover the defect site, preventing graft dislodgement. The skin was sutured, and the rabbit was returned to the animal facility for continued rearing after full recovery. For three consecutive days post-surgery, the rabbits received intramuscular injections of penicillin (provided by the Air Force Medical University Animal Center) at 20,000 units / kg body weight to prevent infection. Four weeks post-surgery, the experimental rabbits were euthanized using the air embolization method. The injured and healthy tibias were removed and fixed in 4% paraformaldehyde solution.

[0139] 6.2 Evaluation of angiogenesis at the defect site using Microfil vascular perfusion 3 weeks postoperatively

[0140] After anesthetizing the rabbits, the abdomen was prepared, and an incision was made in the midline of the abdomen to open the abdominal cavity. The stomach and intestines were turned to one side to expose the abdominal aorta and inferior vena cava sheaths, which were then freed. A perfusion needle was inserted and fixed through a puncture in the anterior wall of the abdominal aorta. The proximal end of the abdominal aorta was ligated, and the distal end of the inferior vena cava was cut. Preheated heparinized saline (50 IU / mL) was perfused at a rate of 2 mL / min until clear saline flowed from the distal end of the inferior vena cava. 50 mL of prepared Microfil angiography agent (purchased from Flow Tech) was then perfused into the blood vessel through the abdominal aorta at the same rate. Successful perfusion was indicated by the appearance of blue fluid flowing from the inferior vena cava and the lower limbs turning blue. Both tibias were removed and soaked in 4% paraformaldehyde overnight at 4°C. The specimen was removed the next day and fixed for another 2 days. It was then decalcified with 10% EDTA until the bone softened, at which point Micro-CT reconstruction analysis could be performed. Results are as follows... Figure 14 As shown.

[0141] The results showed that multiple blood vessels passed through the injury site in the Blank group, while only a few blood vessels passed through the TSP1- group. +The group with the fewest ingrowth vessels at the defect site was the Blank group. Observing the sagittal section of the tibial specimen perfused with microfil, the Blank group showed more and larger vessels forming at the injury site compared to the two groups with implanted stents. The TSP1- group had fewer ingrowth vessels, while the TSP1 group... + The group had the fewest and smallest blood vessels. This indicates that regardless of whether TSP1 is present, the stent has a certain inhibitory effect on blood vessel ingrowth, and the addition of TSP1 further strengthens this inhibitory effect.

[0142] 6.3 Evaluation of repair effect 8 weeks postoperatively

[0143] 6.3.1 General analysis

[0144] Carefully remove the connective tissue, including muscles, tendons, and ligaments, attached to the tibia on both the injured and healthy sides, taking care not to damage the articular surfaces on both sides. First, draw a tangent line along the proximal articular surface of the injured tibia (right limb tibia). Then, draw a straight line from the center of the proximal and distal articular surfaces. The two lines intersect at the proximal end of the tibia, and the angle on the medial side (opposite to the fibula) is recorded as β. Using the healthy tibia as a reference, draw two lines in the same way, and record the angle at the corresponding position as α. Here, α is the normal angle, generally above 90°. Since the epiphyseal plate injury is located on the medial side of the proximal tibia, the loss of growth potential on the medial side will inevitably lead to a lower medial articular surface, thus making the β angle smaller. Based on this, the difference between the α angle and the β angle is the angle difference between the two tibias, i.e., the angle of angular deformity. In addition, measure the length of the tibia on the injured and healthy sides separately using calipers, accurate to two decimal places. Epiphyseal plate injury, in principle, leads to epiphyseal plate dysfunction, thereby inducing limb shortening. Therefore, the difference between the length of the healthy tibia and the length of the injured tibia is the direct evaluation parameter for tibial shortening, i.e., the difference in tibial length. The results are as follows: Figure 1 As shown in Figure 5.

[0145] The results showed that the Blank group and TSP1 - All groups had severe angular deformities, while in TSP1 + Only one case in the Blank group showed a clearly visible deformity. Statistical results indicate that the Blank group and TSP1... - There was no difference in the degree of angular deformity among the groups, with an average deformity angle of around 30°. However, TSP1... + The degree of deformity in this group was significantly milder than in the other two groups, with an average deformity angle of approximately 10°. The difference in tibial medial and lateral length (average approximately 2 mm) in the developmental growth plate layered scaffold group was closer to normal. (TSP1) + The shortening in the TSP1 group (average shortening of approximately 1.5 mm) was significantly reduced compared to the Blank group (average shortening of approximately 3.5 mm). This indicates that TSP1... + Developmental growth plate layered scaffolds can effectively prevent angular deformities and also have a positive effect on tibial shortening.

[0146] 6.3.2 HE staining

[0147] After decalcification, the tibia was sectioned in the sagittal plane and stained with hematoxylin and eosin (HE). The results are as follows: Figure 16 As shown.

[0148] The results showed that in the Blank group, numerous bone bridges formed at the injury site, and these bridges were continuous and extended into the medullary cavity. In contrast, in the TSP1 group... - The number of bone bridges in the TSP1 group was significantly reduced compared to the blank defect group, and some bone bridges were discontinuous with visible fibrous tissue filling. + The group showed a significant reduction in bony filling at the defect site, with more dispersed bone bridges and a small amount of cartilage tissue. These results indicate that TSP1... - Developmental growth plate composite scaffolds can slightly reduce bone bridge formation, while TSP1 + The developmental growth plate composite scaffold has a stronger effect in preventing bone bridging and also plays a positive role in cartilage regeneration.

[0149] 6.3.3 MicroCT Analysis

[0150] The proximal tibia was scanned using a SkyScan 1276 instrument (Bruker), with a scan resolution of 40 μm, a scan voltage of 70 kV, and a current of 200 μA. Bone bridge selection: Using CTAn, the growth plate lesion site was drawn along the subchondral bone margin on the coronal plane, with a thickness of 3 mm. The volume of this region was measured as the bone bridge volume fraction (BV). Results are as follows... Figure 17 As shown.

[0151] The results showed that the bone bridges formed at the injury sites in the Blank group were denser, and TSP1 - The newly formed bone tissue in this group is high in content and relatively dense, TSP1 + The total amount of bone tissue at the defect sites was significantly reduced, and the structure was relatively porous. Statistical results further confirm that TSP1... + The volume fraction of bone bridges formed in group TSP1 was significantly lower than that in the other two groups. The trabecular pattern factor was used to reflect the degree of trabecular irregularity; the higher the degree of irregularity, the larger the value. + The trabecular pattern factor in the first group was significantly higher than that in the other two groups, indicating that the newly formed bone was more porous. The results of microCT assessment of bone bridges were consistent with those of HE staining.

[0152] In summary, TSP1-mediated angiogenesis inhibition is a key factor in the reduction of bone bridges at defect sites. The present invention prepares a vascular barrier containing TSP1... + The developmental growth plate layered scaffold improves the pathological microenvironment of growth plate defects by inhibiting angiogenesis, making it more suitable for chondrocyte survival. In addition, the excellent anti-teratogenic effect of the layered scaffold also shows great translational value.

[0153] As can be seen from the above embodiments, the present invention provides a developmental growth plate layered scaffold, its preparation method, and its application. The present invention constructs a layered scaffold with developmental growth plate characteristics, and on this basis, simulates the dynamic differentiation order of growth plate chondrocytes and repairs damaged growth plates in situ, thereby preventing or alleviating skeletal deformities. Under physiological conditions, growth plate cells have two key characteristics: first, cells exhibit a dynamic evolutionary pattern of "resting-proliferation-hypertrophy-apoptosis or transdifferentiation," gradually migrating from the upper layer to the metaphysis and participating in bone growth; second, chondrocytes exist in an avascular microenvironment, with blood supply barriers existing in the epiphysis and metaphysis. The design concept of the layered scaffold in this invention is as follows: the upper layer is a chondrocyte layer, where cells simultaneously exist in both proliferative and differentiated states; the lower layer is a calcified layer, which communicates with the bone marrow and is an active osteogenic area; the middle layer is a vascular barrier layer, which can act as an adhesive to bond the upper and lower layers to maintain the structural stability of the multi-layered scaffold, and also as a vascular barrier to prevent blood vessels from growing from the lower layer into the upper layer. In this invention, the layered scaffold only has cells loaded in the upper layer. These cells pass through the middle layer and colonize in the lower layer. This process is similar to the dynamic evolution of cells in a natural growth plate.

[0154] 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 developmental growth plate layered scaffold, characterized in that, It comprises three layers: upper, middle, and lower; the upper layer is a chondrocyte layer, the middle layer is a vascular barrier layer, and the lower layer is an ossification layer. The chondrocyte layer is loaded with bone marrow mesenchymal stem cells; The vascular barrier layer is loaded with platelet-reactive protein 1; The ossified layer is composed of type I collagen and hydroxyapatite; The method for preparing the chondrocyte layer includes the following steps: Bone marrow mesenchymal stem cells with a cell fusion rate of 80-90% were digested and resuspended to obtain a cell resuspension; the cell resuspension was dropped onto a microcarrier, allowed to stand, and induction differentiation medium was added for dispersion treatment. The induction differentiation medium was replaced and incubated to obtain the chondrocyte layer. The method for preparing the vascular barrier layer includes the following steps: Methacrylamide gelatin and photoinitiator were dissolved separately, platelet-reactive protein 1 was added, and the mixture was irradiated with blue light or ultraviolet light to obtain the vascular barrier layer. The method for preparing the ossified layer includes the following steps: Type I collagen was dissolved and hydroxyapatite was added to obtain 3D printing bio-ink. The ink was then added to a 3D bioprinter for printing to obtain a type I collagen / hydroxyapatite scaffold. The type I collagen / hydroxyapatite scaffold was freeze-dried and cross-linked to obtain the ossification layer.

2. The developmental growth plate layered scaffold according to claim 1, characterized in that, In the method for preparing the chondrocyte layer, the density of the cell resuspension is 1~3×10⁻⁶. 6 The concentration of cells / mL is 37°C, the standing temperature is 2-3 hours, the frequency of changing the induction differentiation medium is 2-3 times per day, and the incubation time is 5-7 days.

3. The developmental growth plate layered scaffold according to claim 1, characterized in that, In the method for preparing the vascular barrier layer, a phosphate buffer solution is used for dissolution, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine, and the effective concentration of platelet-reactive protein 1 is 5 µg / mL.

4. The developmental growth plate layered scaffold according to claim 1, characterized in that, In the preparation method of the ossified layer, acetic acid is used for dissolution; the mass ratio of type I collagen to hydroxyapatite is 1~2:1; the freeze-drying time is 4~6h; and genipin is used for cross-linking.

5. A method for assembling a developmental growth plate layered scaffold according to any one of claims 1 to 4, characterized in that, Includes the following steps: The chondrocyte layer was cut into 5×5mm pieces and adhered to the vascular barrier layer to obtain a pre-assembled scaffold. A polyvinylidene fluoride membrane was inserted from one side of the pre-assembled scaffold, and the insertion was stopped at 1 / 2 of the pre-assembled scaffold. The pre-assembled scaffold was then lifted and pushed onto the ossified layer for cross-linking. It was then transferred to a chondrogenic induction culture medium for culture to obtain the developmental growth plate layered scaffold.

6. The assembly method according to claim 5, characterized in that, The crosslinking is performed under a 405nm light source; the crosslinking time is 14~16s; the culture temperature is 37℃; and the culture time is 5~7d.

7. The use of the developmental growth plate layered scaffold according to any one of claims 1 to 4 in the preparation of products for repairing epiphyseal injuries.

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