Controllable gradient scaffold preparation method and controllable gradient scaffold

Abstract

Provided are a controllable gradient scaffold preparation method and a controllable gradient scaffold. Cells are modified by means of mRNA delivery, and the modified cells are loaded to a specific position to construct a gradient scaffold. The controllable gradient scaffold preparation method relates to functionalizing seed cells by means of mRNA transfection, and thus achieves better effectiveness, safety and clinical translation potential than traditional methods involving recombinant proteins, plasmids, metal ions, etc. In addition, the used mRNA is highly designable, such that on the basis of the regeneration needs of different tissues, functional factors required for encoding can be designed, to design specific shapes and internal structures to satisfy clinical needs, thereby achieving personalized structural customization and better satisfying the needs of accurately inducing trilineage differentiation of BMSCs to cartilage, hypertrophic cartilage and bone within a three-dimensional space of the scaffold.

Description

A preparation method of a controllable gradient scaffold and a controllable gradient scaffold 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 defects are a common clinical condition. Arthroplasty, autologous cartilage / chondrocyte transplantation, and total joint replacement are currently common methods for treating 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 cartilage, 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. Bone marrow stromal cells (BMSCs) have the ability to differentiate into these three functional cell types and are the main cells involved in tissue repair after osteochondral injury. Using advanced manufacturing technologies, such as bio-3D printing technology, to create 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 physical and chemical 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 problems such as the diffusion and inactivation of ions and growth factors, it is still difficult to regenerate the multiphase structure of natural osteochondral cartilage. In particular, the lack of a calcified cartilage layer allows 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 inductive microenvironment to meet the needs of accurately inducing BMSCs to differentiate into cartilage, hypertrophic cartilage and osteogenesis within the three-dimensional space of the scaffold. Technical Solutions

[0004] In view of this, it is necessary to provide a preparation method 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 this application is to provide a method for preparing a controllable gradient scaffold, comprising the following steps:

[0007] Cells are modified by mRNA delivery, and the modified cells are loaded into specific locations to construct the gradient scaffold.

[0008] In some embodiments, the mRNA comprises conventional mRNA, self-replicating mRNA, or circular mRNA.

[0009] In some embodiments, the cells may be derived from a single tissue or from multiple tissues.

[0010] In some embodiments, in the modification of cells by mRNA delivery, the delivery can be single mRNA delivery or co-delivery of multiple mRNAs.

[0011] In some embodiments, the mRNA delivery carrier can be a liposome, or a polymer, exosome, extracellular vesicle, 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 into specific locations by 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] mRNA delivery was used to transfect mRNA encoding chondrogenic and osteogenic proteins into cells respectively;

[0016] The mRNA-modified cells were collected and uniformly mixed with the hydrogel to prepare osteogenic printing ink and chondrogenic printing ink, respectively;

[0017] 3D printing is performed using the osteogenic printing ink and the chondrogenic printing ink as printing inks, and the modified cells are loaded into specific positions to construct a gradient scaffold.

[0018] In some embodiments, the controllable gradient scaffold may be a single layer or multiple layers, and its gradient may be a single gradient or multiple gradients.

[0019] The second purpose of this application is to provide a controllable gradient scaffold prepared by the controllable gradient scaffold preparation method. Beneficial effects

[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 the modified cells are loaded into 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 appearance 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 following briefly introduces the drawings required for use in the embodiments of the present application or the description of the prior art. 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 any creative work.

[0023] FIG1 shows the highly expressed Runx2 transcription factor in the gene-functionalized BMSC cells provided in Examples 2 and 3 of the present application.

[0024] FIG. 2 shows the highly expressed Sox9 transcription factor in the gene-functionalized BMSC cells provided in Examples 2 and 3 of the present application.

[0025] Figure 3 shows a two-phase gradient scaffold loaded with gene-functionalized cells 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 shows a three-phase gradient scaffold loaded with gene-functionalized cells 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 shows that the two-phase gradient scaffold loaded with mRNA functionalized cells provided in Example 2 of the present application promotes the repair of rabbit articular osteochondral defects (results 3 months after surgery). Modes for Carrying Out the Invention

[0028] The following describes in detail embodiments of the present application, examples of which 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 this 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 this 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 this application.

[0030] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0031] In order to make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below with reference to the accompanying drawings and embodiments.

[0032] The method for preparing a controllable gradient scaffold provided in the present embodiment includes: modifying cells by mRNA delivery, and loading the modified cells into specific locations 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 is understood that the present application utilizes mRNA transfection to functionalize seed cells, which has better efficacy, safety, and clinical translation potential than traditional methods such as recombinant proteins, plasmids, and metal ions. Furthermore, because the mRNA used is highly designable, it can be designed to encode the desired functional factors based on the regeneration needs of different tissues.

[0035] Furthermore, the mRNA used in this embodiment can be replaced with 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 derived from a single tissue or from multiple tissues.

[0037] It is understood that the cells described in this embodiment can be bone marrow stromal cells (BMSCs) or can be replaced by other stem cells or precursor cells with differentiation potential, such as adipose-derived MSCs, induced pluripotent stem cells, and chondrogenic progenitor cells; they can also be adult cells such as chondrocytes, osteoblasts, and endothelial cells.

[0038] In this embodiment, in the modification of cells by mRNA delivery, the delivery can be single mRNA delivery or co-delivery of multiple mRNAs.

[0039] Furthermore, the mRNA delivery carrier can be a liposome, or a polymer, exosome, extracellular vesicle, cell membrane, or electroporation.

[0040] In this embodiment, the step of loading the modified cells to a specific position to construct a gradient scaffold is specifically as follows: loading the modified cells to a specific position to construct a gradient scaffold by 3D printing, gas foaming, pore-forming agent precipitation, or electrospinning.

[0041] It is understood 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 several 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, the step of loading the modified cells to a specific position by 3D printing to construct a gradient scaffold specifically includes the following steps:

[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: performing 3D printing using the osteogenic printing ink and the chondrogenic printing ink as printing inks, and loading the modified cells 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 with different interface structures, achieving refined bionics of the physiological bone and cartilage structure.

[0048] It is 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, and 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 appearance 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.

[0051] The following is a detailed description of a controllable gradient scaffold loaded with mRNA functionalized cells, its preparation method and application in conjunction with specific implementation cases.

[0052] Example 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 (Sox9 mRNA), and a UV crosslinker to prepare cartilage printing material. The concentration of GelMA was 5-15% (mass-volume ratio), the amount of Sox9 mRNA was 2.5-25 pg / cell, and the BMSC content was 1×10 6 ~5×10 6 / ml, the concentration of UV crosslinker is 0.05%~0.3%;

[0054] B. Subchondral bone printing material was prepared by uniformly mixing double-bond modified gelatin (GelMA), bone marrow mesenchymal stem cells (BMSC-Runx2) transfected with messenger RNA encoding Runx2 transcription factor (Runx2 mRNA), and a UV crosslinker. The concentration of GelMA was 5-15% (mass-volume ratio), the amount of Runx2 mRNA was 2.5-25 pg / cell, and the BMSC content was 1×10 6 ~5×10 6 / ml, the concentration of UV crosslinker is 0.05%~0.3%;

[0055] C. The printing process uses a multi-nozzle alternating printing method: first, the subchondral bone layer is built. After reaching the set height, the print head is switched to print the cartilage layer;

[0056] D. After the gradient scaffold is printed, use a UV flashlight or UV crosslinking box to cure the scaffold;

[0057] E. The obtained scaffold can be added to the culture medium and cultured in a 37 ℃ incubator for in vitro research

[0058] F. For in vivo studies, direct implantation is performed.

[0059] Example 2

[0060] A. Runx2 and Sox9 mRNAs were first transfected into BMSCs at 10 pg / cell. Figures 1 and 2 show that 24 hours after transfection, Runx2 and Sox9 transcription factor expression in BMSCs was significantly upregulated.

[0061] B. BMSC-Runx2 cells were cultured at 5×10 6 / ml concentration was evenly mixed with GelMA solution (10% w / v) to prepare the subchondral bone layer printing material;

[0062] C. BMSC-Sox9 cells were cultured at 5×10 6 / ml concentration was evenly mixed with GelMA solution (10% w / v) to prepare the cartilage layer printing material;

[0063] D. The obtained printing materials were loaded into the material barrels respectively, and the printing needles with an inner diameter of 350 μl were installed, and then alternate printing was performed.

[0064] As shown in Figure 3, the cartilage layer was printed first, with a total of four layers. Six layers of subchondral bone were then printed above the cartilage layer. After printing, the scaffold was cross-linked and cured using a UV lamp for 45 seconds, ultimately resulting in a two-phase gradient scaffold containing two functionalized cell types.

[0065] E. The resulting gradient scaffold was implanted into a rabbit femoral condyle osteochondral defect. Three months later, magnetic resonance imaging (MRI) revealed that the gradient scaffold containing mRNA-functionalized cells significantly promoted the regeneration and repair of the defect (Figure 5).

[0066] Example 3

[0067] A. Runx2 and Sox9 mRNA were first transfected into BMSCs at 10 pg / cell. Figures 1 and 2 show that 24 hours after transfection, the expression of Runx2 and Sox9 transcription factors in BMSCs was significantly upregulated.

[0068] B. BMSC-Runx2 cells were cultured at 5×10 6 The concentration of PDMS / PDMS was uniformly 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 The concentration of PDMS / PDMS was uniformly mixed with GelMA solution (10% w / v) to prepare the cartilage layer printing material.

[0070] D. The obtained printing materials were loaded into the material barrels respectively, and the printing needles with an inner diameter of 350 μl were installed, and then alternate printing was performed.

[0071] As shown in Figure 4, the cartilage layer was printed first, with a total of two layers. The two materials were then alternately printed on top, for a total of four layers. Finally, four layers of subchondral bone were printed. After printing, the scaffold was cross-linked and cured using a UV lamp for 45 seconds, resulting in a three-phase gradient scaffold containing two functionalized cells.

[0072] It can be understood that the various technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the various 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 merely preferred embodiments of the present application and only specifically describe the technical principles of the present application. These descriptions are intended only to explain the principles of the present application and should not be construed in any way as limiting the scope of protection of the present application. Based on the explanations herein, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present application, as well as other specific implementations of the present application that can be conceived by those skilled in the art without inventive effort, shall be included within 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.

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