Preparation method of controllable gradient stent and controllable gradient stent
The cells are modified through mRNA delivery and a controlled gradient scaffold is solved, which is difficult to achieve precise differentiation of BMSCs in the prior art, and improves the success rate of repair of joint osteocartilage defects.
Patent Information
- Application Number
- CN202311221438.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-20
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art is difficult to achieve precisely inducing BMSCs to differentiate into the three-dimensional system of cartilage, hypertrophic cartilage and osteogenic system in the treatment of joint osteocartilage defects, resulting in a low repair success rate.
The cells are modified through mRNA delivery, and the modified cells are loaded to a specific position to build a controllable gradient scaffold, and the preparation of gradient scaffolds is achieved using technologies such as 3D printing.
The precise induction of BMSCs in the three-dimensional space of the stent was achieved to differentiate BMSCs into cartilage, hypertrophic cartilage and osteogenesis, and the success rate of repair of joint osteocartilage defects was improved.
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Figure CN119662739A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of biomedical engineering technology, and in particular to a method for preparing a controllable gradient scaffold and the controllable gradient scaffold. Background Art
[0002] Articular osteochondral defect is a common clinical disease. Arthroplasty, autologous cartilage / chondrocyte transplantation, total joint replacement and other methods are currently commonly used to treat osteochondral defects. Although these methods can effectively improve symptoms in the short term, it is difficult to completely restore the natural structure and function of osteochondral, resulting in a low success rate of repair. The formation and maintenance of the cartilage layer-calcified cartilage layer-subchondral bone layer gradient structure of the articular osteochondral unit mainly rely on the participation of chondrocytes, hypertrophic chondrocytes and osteoblasts, while bone marrow stromal cells (BMSCs) have the ability to differentiate into these three functional cells and are the main cells involved in tissue repair after osteochondral injury. Using advanced manufacturing technologies, such as biological 3D printing technology, creating an integrated osteochondral regeneration scaffold with an osteochondral gradient structure uniformly loaded with BMSCs provides a new strategy for the treatment of articular osteochondral defects.
[0003] In order to achieve the functionalization of BMSCs in the scaffold, researchers have constructed a variety of osteochondral repair scaffolds with physicochemical induced gradients: by imitating the extracellular matrix composition, hardness or pore size / porosity differences of osteochondral units, or introducing a variety of growth factors, metal ions, etc., to promote the directional differentiation of BMSCs. However, due to the harsh conditions for the preparation of physical gradients, as well as the diffusion and inactivation of ions and growth factors, it is still difficult to regenerate the multiphase structure of natural osteochondral. In particular, the lack of a calcified cartilage layer causes the subchondral bone to invade the cartilage, resulting in cartilage calcification and vascularization, and ultimately causing the failure of osteochondral defect repair. Therefore, researchers need to explore the construction of a new induced microenvironment to meet the needs of accurately inducing BMSCs to differentiate into cartilage, hypertrophic cartilage and osteogenesis in the three-dimensional space of the scaffold. Summary of the invention
[0004] In view of this, it is necessary to provide a method for preparing a controllable gradient scaffold and a controllable gradient scaffold that can meet the needs of accurately inducing BMSCs to differentiate into cartilage, hypertrophic cartilage and osteogenesis in the three-dimensional space of the scaffold to address the defects of the existing technology.
[0005] To solve the above problems, this application adopts the following technical solutions:
[0006] One of the purposes of the present application is to provide a method for preparing a controllable gradient scaffold, comprising the following steps:
[0007] The cells are modified by mRNA delivery, and the modified cells are loaded to a specific location to construct the gradient scaffold.
[0008] In some of these embodiments, the mRNA includes conventional mRNA or self-replicating mRNA or circular mRNA.
[0009] In some embodiments, the cells may be derived from a single tissue or may be derived from multiple tissues.
[0010] In some of the embodiments, in the modification of cells by mRNA delivery, the delivery may be a single mRNA delivery or a co-delivery of multiple mRNAs.
[0011] In some of the embodiments, the mRNA delivery carrier can be a liposome, or a polymer, an exosome, an extracellular vesicle, a cell membrane, or electroporation.
[0012] In some embodiments, in the step of loading the modified cells to a specific location to construct a gradient scaffold, specifically:
[0013] The modified cells are loaded to specific locations through 3D printing, gas foaming, pore-forming agent precipitation, or electrospinning to construct a gradient scaffold.
[0014] In some embodiments, the step of loading the modified cells to a specific location by 3D printing to construct a gradient scaffold specifically includes the following steps:
[0015] mRNAs encoding chondrogenic and osteogenic proteins were transfected into cells respectively using mRNA delivery;
[0016] The mRNA-modified cells are collected and uniformly mixed with the hydrogel to prepare osteogenic printing ink and chondrogenic printing ink, respectively;
[0017] The osteogenic printing ink and the chondrogenic printing ink are used as printing inks for 3D printing, and the modified cells are loaded to specific locations to construct a gradient scaffold.
[0018] In some embodiments, the controllable gradient support may be one layer or multiple layers, and its gradient may be a single gradient or multiple gradients.
[0019] The second purpose of the present application is to provide a controllable gradient scaffold prepared by the controllable gradient scaffold preparation method.
[0020] This application adopts the above technical solution, and its beneficial effects are as follows:
[0021] The preparation method of the controllable gradient scaffold and the controllable gradient scaffold provided in the present application utilize mRNA delivery to modify cells, and load the modified cells to a specific position to construct the gradient scaffold. The preparation method of the controllable gradient scaffold provided in the present application utilizes mRNA transfection to functionalize seed cells, which has better effectiveness, safety and clinical transformation potential than traditional recombinant proteins, plasmids, metal ions and the like; at the same time, the mRNA used has extremely high designability, and can be designed to encode the required functional factors according to the regeneration needs of different tissues, so as to design a specific shape and internal structure to meet clinical needs, realize personalized customization of the structure, and better meet the needs of accurately inducing BMSCs to differentiate into cartilage, hypertrophic cartilage and osteogenesis in the three-dimensional space of the scaffold. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0023] Figure 1 The Runx2 transcription factor is highly expressed in the gene-functionalized BMSC cells provided in Examples 2 and 3 of the present application.
[0024] Figure 2 The Sox9 transcription factor is highly expressed in the gene-functionalized BMSC cells provided in Examples 2 and 3 of the present application.
[0025] Figure 3 The two-phase gradient scaffold loaded with gene-functionalized cells was constructed by biological 3D printing provided in Example 2 of the present application (the green area contains BMSC-Sox9 cells, and the red area contains BMSC-Runx2 cells).
[0026] Figure 4 The three-phase gradient scaffold loaded with gene-functionalized cells was constructed by biological 3D printing provided in Example 3 of the present application (the green area contains BMSC-Sox9 cells, and the red area contains BMSC-Runx2 cells).
[0027] Figure 5 The two-phase gradient scaffold loaded with mRNA functionalized cells provided in Example 2 of the present application promoted the repair of osteochondral defects in rabbit joints (results 3 months after surgery). DETAILED DESCRIPTION
[0028] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.
[0029] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.
[0030] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.
[0031] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.
[0032] The preparation method of the controllable gradient scaffold provided in the embodiment of the present application includes: modifying cells by mRNA delivery, and loading the modified cells to a specific position to construct the gradient scaffold. The implementation method is described in detail below.
[0033] In this embodiment, the mRNA includes conventional mRNA, self-replicating mRNA or circular mRNA.
[0034] It can be understood that the present application uses mRNA transfection to functionalize seed cells, which has better effectiveness, safety and clinical transformation potential than traditional recombinant proteins, plasmids, metal ions, etc. And because the mRNA used has extremely high designability, it can be designed to encode the required functional factors according to the regeneration needs of different tissues.
[0035] Furthermore, the mRNA used in this embodiment can be replaced by mRNA encoding other desired functional factors, such as transforming growth factor β1, 3 (TGFβ1, 3), bone morphogenetic protein 2, 7, 9 (BMP2, 7, 9), vascular endothelial growth factor (VEGF mRNA), nerve growth factor (NGF mRNA), etc.
[0036] In this embodiment, the cells may be cells derived from a single tissue or cells derived from multiple tissues.
[0037] It is understood that the cells described in this embodiment can be bone marrow stromal cells BMSC or can be replaced by other stem cells or precursor cells with differentiation potential, such as adipose-derived MSC, induced pluripotent stem cells, chondrogenic progenitor cells; they can also be adult cells such as chondrocytes, osteoblasts, endothelial cells, etc.
[0038] In this embodiment, in the modification of cells by mRNA delivery, the delivery can be a single mRNA delivery or a co-delivery of multiple mRNAs.
[0039] Furthermore, the mRNA delivery carrier may be a liposome, or may be a polymer, exosome, extracellular vesicle, cell membrane, or electroporation.
[0040] In this embodiment, in the step of loading the modified cells to a specific position to construct a gradient scaffold, specifically: loading the modified cells to a specific position to construct a gradient scaffold by 3D printing or gas foaming or pore-forming agent precipitation method or electrospinning.
[0041] It is understandable that the cells can be inoculated into the scaffold after the scaffold is prepared, or can be introduced simultaneously during the scaffold preparation process.
[0042] It should be noted that the number of layers of the constructed controllable gradient stent can be a few layers or a multi-layer stent, and its gradient can be a single gradient or a complex gradient, depending on actual clinical needs.
[0043] Furthermore, in the step of loading the modified cells to a specific position by 3D printing to construct a gradient scaffold, the following steps are specifically included:
[0044] S110: mRNA encoding chondrogenic and osteogenic proteins were transfected into cells using mRNA delivery;
[0045] Step S120: collecting the mRNA-modified cells and uniformly mixing them with the hydrogel to prepare osteogenic printing ink and chondrogenic printing ink respectively;
[0046] Step S130: 3D printing is performed using the osteogenic printing ink and the chondrogenic printing ink as printing inks, and the modified cells are loaded to specific locations to construct a gradient scaffold.
[0047] Specifically, this embodiment adopts a multi-nozzle alternating printing method to construct an integrated bone and cartilage scaffold printing with different interface structures, thereby achieving refined bionics of the physiological bone and cartilage structure.
[0048] It can be understood that in practical applications, the obtained gradient scaffold is implanted into the osteochondral defect of experimental animals to examine its ability to promote tissue regeneration.
[0049] It should be noted that the shape of the controllable gradient scaffold loaded with mRNA functionalized cells is related to the designed printing model. The printing model can be designed according to different needs to print three-dimensional scaffolds with different morphologies, sizes or gradients, or even complex organs.
[0050] The preparation method of the controllable gradient scaffold and the controllable gradient scaffold provided above in the present application utilize mRNA delivery to modify cells, and load the modified cells to a specific position to construct the gradient scaffold. The preparation method of the controllable gradient scaffold provided in the present application utilizes mRNA transfection to functionalize seed cells, which has better effectiveness, safety and clinical transformation potential than traditional recombinant proteins, plasmids, metal ions and the like; at the same time, the mRNA used has extremely high designability, and can be designed to encode the required functional factors according to the regeneration needs of different tissues, so as to design a specific shape and internal structure to meet clinical needs, realize personalized customization of the structure, and better meet the needs of accurately inducing BMSCs to differentiate into three lines of cartilage, hypertrophic cartilage and osteogenesis in the three-dimensional space of the scaffold.
[0051] The following is a further detailed description of a controllable gradient scaffold loaded with mRNA functionalized cells and its preparation method and application according to the present invention in conjunction with specific implementation examples.
[0052] Embodiment 1:
[0053] A. Double-bond modified gelatin (GelMA) was uniformly mixed with bone marrow mesenchymal stem cells (BMSC-Sox9) transfected with messenger RNA encoding Sox9 transcription factor (Sox9mRNA) and a UV crosslinker to prepare cartilage printing material. The concentration of GelMA was 5-15% (mass-volume ratio), the amount of Sox9mRNA was 2.5-25pg / cell, and the content of BMSC was 1×10 6 ~5×10 6 / ml, the concentration of UV crosslinker is 0.05% to 0.3%;
[0054] B. Double-bond modified gelatin (GelMA) was uniformly mixed with bone marrow mesenchymal stem cells (BMSC-Runx2) transfected with messenger RNA encoding Runx2 transcription factor (Runx2 mRNA) and a UV crosslinker to prepare subchondral bone printing material; the concentration of GelMA was 5-15% (mass-volume ratio), the amount of Runx2 mRNA was 2.5-25 pg / cell, and the content of BMSC was 1×10 6 ~5×10 6 / ml, the concentration of UV crosslinker is 0.05% to 0.3%;
[0055] C. The printing process uses a multi-nozzle alternating printing method: first, the subchondral bone layer is constructed, and after reaching the set height, the print nozzle is switched to print the cartilage layer;
[0056] D. After the gradient bracket is printed, use a UV flashlight or UV cross-linking box to cure the bracket;
[0057] E. The obtained scaffold can be added to the culture medium and cultured in a 37°C incubator for in vitro research
[0058] F. For in vivo studies, direct in vivo implantation is performed.
[0059] Example 2
[0060] A. First, Runx2 mRNA and Sox9 mRNA were transfected into BMSC cells at a concentration of 10 pg / cell. Figure 1 and Figure 2 It showed that 24h after transfection, the expression of Runx2 and Sox9 transcription factors in BMSC cells was significantly upregulated;
[0061] B. BMSC-Runx2 cells were cultured at 5×10 6 / ml concentration was uniformly mixed with GelMA solution (10% w / v) to prepare subchondral bone layer printing material;
[0062] C. BMSC-Sox9 cells were cultured at 5×10 6 / ml concentration was uniformly mixed with GelMA solution (10% w / v) to prepare the cartilage layer printing material;
[0063] D. Load the obtained printing materials into the barrels respectively, install the printing needles with an inner diameter of 350μl, and then perform alternate printing.
[0064] like Figure 3 As shown, the cartilage layer is printed first, with a total of 4 layers, and then 6 layers of subchondral bone are printed on the cartilage layer. After printing, the scaffold is cross-linked and cured for 45 seconds using an ultraviolet lamp, and finally a two-phase gradient scaffold containing two functional cells is obtained.
[0065] E. The obtained gradient scaffold was implanted into the osteochondral defect of the rabbit femoral condyle. Magnetic resonance imaging (MRI) was used 3 months later to find that the gradient scaffold containing mRNA functionalized cells better promoted the regeneration and repair of defective tissue ( Figure 5 ).
[0066] Example 3
[0067] A. First, Runx2 mRNA and Sox9 mRNA were transfected into BMSC cells at a concentration of 10 pg / cell. Figure 1 and Figure 2 The results showed that 24h after transfection, the expression of Runx2 and Sox9 transcription factors in BMSC cells was significantly upregulated.
[0068] B. BMSC-Runx2 cells were cultured at 5×10 6 The concentration of 1:100 μg / ml was evenly mixed with GelMA solution (10% w / v) to prepare the subchondral bone layer printing material.
[0069] C. BMSC-Sox9 cells were cultured at 5×10 6 / ml concentration was evenly mixed with GelMA solution (10% w / v) to prepare cartilage layer printing material.
[0070] D. Load the obtained printing materials into the barrels respectively, install the printing needles with an inner diameter of 350μl, and then perform alternate printing.
[0071] like Figure 4 As shown, the cartilage layer is printed first, with a total of 2 layers, and then the two materials are printed alternately on top, with a total of 4 layers, and finally 4 layers of subchondral bone are printed. After printing, the scaffold is cross-linked and cured for 45 seconds using an ultraviolet lamp to obtain a three-phase gradient scaffold containing two functional cells.
[0072] It can be understood that the technical features of the above-described embodiments can be arbitrarily combined. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0073] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.
Claims
1. A method for preparing a controllable gradient scaffold, characterized in that: The steps include: The cells are modified by mRNA delivery, and the modified cells are loaded to a specific location to construct the gradient scaffold.
2. The method for preparing a controllable gradient scaffold according to claim 1, characterized in that: The mRNA includes conventional mRNA, self-replicating mRNA or circular mRNA.
3. The method for preparing a controllable gradient scaffold according to claim 1, characterized in that: The cells may be derived from a single tissue or from multiple tissues.
4. The method for preparing a controllable gradient scaffold according to claim 1, characterized in that: In the modification of cells by mRNA delivery, the delivery can be single mRNA delivery or co-delivery of multiple mRNAs.
5. The method for preparing a controllable gradient scaffold according to claim 4, characterized in that: The carrier for mRNA delivery can be a liposome, or a polymer, exosome, extracellular vesicle, cell membrane, or electroporation.
6. The method for preparing a controllable gradient scaffold according to claim 1, characterized in that: In the step of loading the modified cells to a specific location to construct a gradient scaffold, specifically: The modified cells are loaded to specific locations through 3D printing, gas foaming, pore-forming agent precipitation, or electrospinning to construct a gradient scaffold.
7. The method for preparing a controllable gradient scaffold according to claim 6, characterized in that: The step of loading the modified cells to a specific position by 3D printing to construct a gradient scaffold specifically includes the following steps: mRNAs encoding chondrogenic and osteogenic proteins were transfected into cells respectively using mRNA delivery; The mRNA-modified cells are collected and uniformly mixed with the hydrogel to prepare osteogenic printing ink and chondrogenic printing ink, respectively; The osteogenic printing ink and the chondrogenic printing ink are used as printing inks for 3D printing, and the modified cells are loaded to specific locations to construct a gradient scaffold.
8. The method for preparing a controllable gradient scaffold according to claim 1, characterized in that: The controllable gradient support may be one layer or multiple layers, and its gradient may be a single gradient or multiple gradients.
9. A controllable gradient support, characterized in that: The controllable gradient scaffold is prepared by the preparation method of any one of claims 1 to 8.