Living plant cell-animal cell composite scaffold as well as preparation method and application thereof

Abstract

The invention relates to a living plant cell-animal cell composite scaffold as well as a preparation method and application thereof. The living plant cell-animal cell composite scaffold comprises a 3D printing hydrogel scaffold used as a muscle repair carrier and a 3D printing biological ceramic scaffold used as a bone repair carrier, wherein the 3D printing hydrogel scaffold and the 3D printing biological ceramic scaffold are assembled by simulating a muscle-skeleton structure; the hydrogel scaffold comprises a polymer matrix component, and functional plant cell components, namely diatom cells and mammalian cell components, namely muscle stem cells, which are distributed in the polymer matrix component, the biological ceramic scaffold comprises a biological ceramic scaffold matrix component and mammalian cell component bone marrow mesenchymal stem cells inoculated and loaded on the biological ceramic scaffold matrix component.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomaterial preparation, and particularly relates to a living plant cell-animal cell composite scaffold and a preparation method and application thereof. BACKGROUND

[0002] Fracture is a very common type of orthopedic disease in clinic. Fracture involves multiple tissue composite injuries, such as volumetric muscle loss, soft tissue injury and arterial injury. The fracture healing process is closely related to the surrounding muscles, soft tissues and the like. At present, the biomaterials used for bone injury repair mainly include inorganic ions (silicon, lithium, magnesium, calcium and the like), hydrogels and 3D scaffolds and the like, which realize bone defect repair under the conditions of no growth factor and exogenous cells; the main ones used for skeletal muscle injury repair are sodium alginate hydrogel, hyaluronic acid, polyvinyl alcohol and the like, which can stabilize the defect volume, fill the gap, facilitate cell contact, and are supplemented with electric field, magnetic field and the like to induce directional arrangement repair.

[0003] Skeletal muscle and bone are both highly vascularized tissues with high oxygen consumption rate and strong metabolic activity, and the muscle-bone system is exposed to an oxidative microenvironment at all times, and hypoxia will impair the muscle-bone formation capacity. Studies have shown that the fracture site covered with muscle flaps heals faster and has less infection than the uncovered fracture, indicating that there is cross-talk between the bone-muscle, and the muscle is the "secondary periosteum" of bone repair. Traditional muscle-bone complex injury adopts massage reduction or surgical fixation, and recent studies use hydrogel scaffolds to deliver growth factors to realize the combined treatment of bone defect and volumetric muscle loss. However, these means have problems such as single action on tissues, central structure hypoxia, limited oxygen production of inorganic materials, expensive cost of cytokines, and lack of discussion on the cross-talk between muscle and bone.

[0004] Living microalgae material is a research hotspot emerging in recent years, and through advanced engineering methods such as component extraction, surface modification, material compounding, genetic engineering and additive manufacturing, the living microalgae material can be used as a construction body for drug delivery, tissue repair and organ model. Diatom is a kind of near-shore planktonic unicellular microalgae, and the shell is a porous silica cell wall (i.e. cone), which has species specificity and morphology diversity. Diatom has photosynthetic pigments and can be autotrophic through photosynthesis. Diatom has broad prospects in biomedicine and has excellent biocompatibility, and appropriate concentration of silicon ions can stimulate osteogenic gene expression and protein secretion, and has potential positive effects on the repair of various bone and bone-related cells and tissues. By using living diatom and combining advanced biological manufacturing technology, an engineered tissue is constructed to provide clues for complex sports medicine injury repair. SUMMARY

[0005] In view of the above technical problems, the present application aims to provide a living plant cell-animal cell composite scaffold and a preparation method and application thereof.

[0006] In a first aspect, the present application provides a living plant cell-animal cell composite scaffold, which comprises a 3D-printed hydrogel scaffold as a muscle repair carrier and a 3D-printed bioceramic scaffold as a bone repair carrier assembled in a manner simulating a "muscle-skeleton" structure; wherein, The hydrogel scaffold comprises a polymer matrix component and a functional plant cell component diatom cell and a mammalian cell component muscle stem cell distributed in the polymer matrix component; The bioceramic scaffold comprises a bioceramic scaffold matrix component and a mammalian cell component bone marrow mesenchymal stem cell inoculated and loaded on the bioceramic scaffold matrix component.

[0007] Preferably, the polymer matrix component comprises methacrylated gelatin; the functional plant cell component diatom cell comprises a chlorella cell, and the bioceramic matrix component comprises β-tricalcium phosphate.

[0008] Preferably, in the hydrogel scaffold, the mass concentration of the polymer matrix component can be 4-8%, preferably 6%; the concentration of the functional plant cell component diatom cell is (0.1-10) × 10 6 / mL, preferably 1 × 10 6 / mL; and the concentration of the mammalian cell component muscle stem cell is (2-4) × 10 6 / mL. In the bioceramic scaffold, the inoculation concentration of the mammalian cell component bone marrow mesenchymal stem cell is (0.5-2) × 10 5 / mL, preferably 1 × 10 5 / mL.

[0009] In a second aspect, the present application provides a preparation method of the above-mentioned living plant cell-animal cell composite scaffold, which comprises the following steps: (1) mixing the functional plant cell component diatom and the mammalian cell component muscle stem cell respectively with a polymer matrix component hydrogel solution to obtain a diatom-based pre-gel bio-ink and a muscle stem cell-based pre-gel bio-ink, and then performing a first 3D printing after pre-cooling to obtain a cross-species symbiotic hydrogel scaffold with diatoms and muscle stem cells; (2) mixing a bioceramic scaffold matrix component with a photocurable resin to obtain a precursor slurry, and then performing a second 3D printing to obtain a bioceramic scaffold matrix after curing and sintering; and then inoculating and loading the mammalian cell component bone marrow mesenchymal stem cell on the bioceramic scaffold matrix to obtain the bioceramic scaffold; (3) The hydrogel scaffold is assembled with the bioceramic scaffold by simulating a "muscle-skeleton" structure to obtain the living plant cell-animal cell composite scaffold.

[0010] Preferably, in step (1), the preparation method of the high-molecular matrix component hydrogel solution comprises: heating to dissolve the methacrylated gelatin in water in a 55-60℃ water bath in the dark, adding a photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and filtering to obtain the solution.

[0011] Preferably, in step (1), the process of the first 3D printing comprises: first depositing 1-6mm or 5-30 layers of the muscle stem cell-based pre-gel biological ink, then depositing 1-6mm or 5-30 layers of the diatom-based pre-gel biological ink, and performing blue light irradiation treatment after layer-by-layer accumulation to form a two-section layered cross-species scaffold.

[0012] Preferably, in step (2), the preparation process of the precursor slurry comprises: mixing 20-45g of β-tricalcium phosphate powder with 25-55g of photocurable resin, placing in a ball mill for 1-3h, and filtering to obtain the slurry.

[0013] Preferably, in step (2), the parameters of the second 3D printing comprise: an exposure time of 5-8s per 40-70μm layer thickness, preferably 6s per 50μm layer thickness.

[0014] Preferably, in step (2), the sintering temperature is 1000-1200℃, and the sintering time is 2-4h, preferably sintering at 1150℃ for 3h.

[0015] In a third aspect, the present application provides a use of the above-mentioned living plant cell-animal cell composite scaffold in the field of muscle-skeleton injury repair.

[0016] Advantages (1) In the living plant cell-animal cell composite scaffold provided by this invention, the living plant cell-animal cell cross-species composite scaffold mixes Chaetoceros (diatoms) and muscle stem cells with methacrylamide gelatin to prepare bio-inks, and then uses 3D bioprinting of living plant cell-animal cell hydrogel scaffolds as muscle repair carriers. This living plant cell-animal cell hydrogel scaffold has good photosynthetic oxygen production characteristics and silicon ion release performance, realizing the in vitro myogenic differentiation of muscle stem cells; β-tricalcium phosphate bioceramic scaffolds are printed by 3D photopolymerization and loaded with bone marrow mesenchymal stem cells as bone repair carriers; simulating the "muscle-skeleton" structure, the pre-myogenic hydrogel scaffold acclimated to the Chaetoceros scaffold is assembled with the bioceramic scaffold loaded with bone marrow mesenchymal stem cells to form a composite scaffold, realizing the in vitro osteogenic differentiation of bone marrow mesenchymal stem cells. The assembly of the pre-myogenic hydrogel scaffold and the bioceramic scaffold promotes the repair of complex muscle-skeleton tissue damage in vivo; (2) The living plant cell-animal cell composite scaffold provided by the present invention can be used as a multifunctional biomaterial to realize the photosynthesis of living diatoms and the differentiation induced by inorganic ions in a cross-species structure, further domesticating the regeneration of surrounding tissues and effectively solving the problem of complex sports medicine injury repair. Attached Figure Description

[0017] Figure 1 The physicochemical properties of the β-tricalcium phosphate bioceramic scaffold 3D printed based on digital light processing in Example 1 are as follows: (a) Optical photograph of the β-tricalcium phosphate bioceramic scaffold; (b) SEM image of the β-tricalcium phosphate bioceramic scaffold; (c) XRD pattern of the β-tricalcium phosphate bioceramic scaffold; (d) Compressive strength-displacement curve of the β-tricalcium phosphate bioceramic scaffold; (e) Theoretical and measured values ​​of porosity of the β-tricalcium phosphate bioceramic scaffold. Figure 2 The basic characteristics of Chaetoceros in Example 1 and the scaffold assembly are shown below: (a) Laboratory culture of Chaetoceros; (b) SEM image of Chaetoceros; (c) UV-vis spectrum of Chaetoceros; (d) Photograph of Chaetoceros scaffold; (e) Dissolved oxygen content change curve of Chaetoceros hydrogel scaffold at 25°C or 37°C and 5000 lx LED irradiation; (f) Cumulative release curve of silicon ions of Chaetoceros hydrogel scaffold at 37°C; (g) Photograph of the nested assembly of methacrylamide hydrogel scaffold and β-tricalcium phosphate bioceramic scaffold. Figure 3 Biocompatibility of the live plant-animal cell cross-species hydrogel scaffold in Example 1: Muscle stem cells and Chaetoceros cells were cultured for 7 days at ratios of (a) 2:1, (b) 3:1 and (c) 4:1. Fluorescent images of calcein AM / PI staining of muscle stem cells in the scaffold. Figure 4Gene expression levels of living plant cell-animal cell cross-species hydrogel scaffold in Example 1: (a) expression of muscle regeneration key factors (MYOD) and stem cell marker genes (PAX7) produced by muscle stem cells, and (b) expression of bone induction factors (OGN and FAM5C) produced by muscle stem cells after the living plant cell-animal cell cross-species hydrogel scaffold was cultured for 7 days; Figure 5 Myogenic differentiation of acerophyte domesticated muscle stem cells in the living plant cell-animal cell cross-species hydrogel scaffold in vitro: (a) is the image of immunofluorescence staining of muscle stem cell skeleton (F-actin) in the scaffold after 7 days of culture; (b) is the image of immunofluorescence staining of muscle stem cell myogenic related proteins (MHC and SCA) in the scaffold after 7 days of culture; Figure 6 Promotion of osteogenic differentiation of bone marrow mesenchymal stem cells by pre-myogenic scaffolds domesticated by acerophyte cross-species in vitro: (a) is the image of immunofluorescence staining of muscle stem cell skeleton (F-actin) loaded in the scaffold after 7 days of culture; (b) is the image of immunofluorescence staining of bone marrow mesenchymal stem cell osteogenic related proteins (OPN and BMP2) loaded in the scaffold after 7 days of culture; Figure 7 Surgical procedure of composite scaffold for rabbit volumetric muscle loss-segmental radial defect in Example 1; Figure 8 Repair effect of composite scaffold for rabbit volumetric muscle loss-segmental radial defect in Example 1-gait analysis: (a) is the representative paw print at 4 and 12 weeks after surgery; (b) is the paw parameter and spatial parameter asymmetry index, including paw length, toe width, step length, stride, step width, at 4 and 12 weeks after surgery; Figure 9 Repair effect of composite scaffold for rabbit volumetric muscle loss-segmental radial defect in Example 1-histological evaluation: (a) is the Micro-CT image and bone volume fraction (BV / TV) value of muscle-radial sample at 4 weeks after surgery; (b) is the Micro-CT image and bone volume fraction (BV / TV) value of muscle-bone sample at 12 weeks after surgery. DETAILED DESCRIPTION

[0018] The present application is further illustrated by the following examples, which are intended to be illustrative only and not limiting of the present application.

[0019] Firstly, the present application provides a living plant cell-animal cell composite scaffold. The living plant cell-animal cell composite scaffold comprises a 3D printed hydrogel scaffold simulating a "muscle-skeleton" structure as a muscle repair carrier and a 3D printed bioceramic scaffold as a bone repair carrier; wherein, The hydrogel scaffold can comprise: a polymer matrix component, and a functional plant cell component diatom (class) cell and a mammalian cell component muscle stem cell distributed in the polymer matrix component. The bioceramic scaffold can comprise: a bioceramic scaffold matrix component, and a mammalian cell component bone marrow mesenchymal stem cell inoculated and loaded in the bioceramic scaffold matrix component.

[0020] In some embodiments, the polymer matrix component can comprise methacrylated gelatin. The polymer matrix used in the present application should have good printability and biocompatibility: (1) temperature sensitivity, flowable in an aqueous solution and cell-friendly temperature range, and achieve sol-gel form transition in the temperature range; (2) good forming performance, i.e. the polymer matrix can be smoothly prepared into uniform filamentous extrudate and complete shape fixation under the action of a pneumatic extrusion printer; (3) good material exchange capacity, sufficient space is needed in the scaffold for the transport and transmission of nutrient components and metabolic products with the culture medium.

[0021] In some embodiments, the functional plant cell component diatom (class) cell can comprise a ceratium (genus) cell.

[0022] It should be noted that the selection of specific species mainly depends on the requirements of experimental parameters. Ceratium is a rich group of coastal bay phytoplankton, which meets the two conditions of releasing silicon ions from siliceous cell walls and containing photosynthetic bodies that can photosynthesize oxygen. In addition, the experiment of the present application is for the physiological environment of mammals (37℃), in addition to the siliceous cell wall and photosynthesis, the selected diatom also needs to meet the condition of survival at 37℃. The suitable survival temperature of common diatoms is 20-30℃, while ceratium can grow and function at 37℃, so ceratium (genus) in diatom (class) is selected as the living diatom material.

[0023] The functional component diatom used in the present application contains chlorophyll and carotenoids in the chloroplast, which can continuously produce oxygen through photosynthesis. In addition, the outer cell wall component of the functional component is a silica shell, which can release silicon ions. The mammalian cell component has differentiation induction potential, and the functional component living diatom used in the present application has domestication effect on the myogenic differentiation of the mammalian cell component muscle stem cell, and the pre-myogenic hydrogel scaffold can effectively accelerate the osteogenic differentiation of the mammalian cell component bone marrow mesenchymal stem cell.

[0024] In some embodiments, the bioceramic matrix component can comprise beta-tricalcium phosphate.

[0025] In some embodiments, the mass concentration (w / v) of the high polymer matrix component in the hydrogel scaffold can be 4-8%, preferably 6%; the concentration of the functional plant cell component diatom cell can be (0.1-10) x 10 6 / mL, preferably 1 x 10 6 / mL; and the concentration of the mammalian cell component muscle stem cell can be (2-4) x 10 6 / mL.

[0026] In the hydrogel scaffold, the concentration of the three components (high polymer matrix, diatom cell, and muscle stem cell) is set to maintain structural stability and ensure cell activity and synergistic effect. If the concentration of the high polymer matrix component is too low, the gel will be too weak to maintain the structure; if the concentration is too high, the pore size will be too small to affect cell migration. The concentration of the diatom cell can ensure the optimal oxygen production effect of photosynthesis at 37℃ and 5000lx. The concentration of the muscle stem cell can ensure cell communication, spatial competition, and nutritional competition.

[0027] In some embodiments, the seeding concentration of the mammalian cell component bone marrow mesenchymal stem cell in the bioceramic scaffold can be (0.5-2) x 10 5 / mL, preferably 1 x 10 5 / mL.

[0028] The concentration of the bone marrow mesenchymal stem cell can ensure the material properties and cell metabolism. If the seeding concentration is too small, the cell proliferation, osteogenic differentiation, and defect repair effect will be poor; if the seeding concentration is too large, the nutrients and living space are limited, and the cell competition is intense. The appropriate concentration is conducive to the effective coverage of the cell on the surface of the bioceramic scaffold, realizes the dynamic balance of nutrients and oxygen, and promotes cell proliferation and osteogenic differentiation.

[0029] The composite scaffold provided by the application has good photosynthetic oxygen production characteristics and silicon ion release performance, realizes the in-vitro myogenic differentiation of muscle stem cells. At the same time, by simulating the "muscle-skeleton" structure, the pre-myogenic hydrogel scaffold domesticated by the scaffold of chlorella oceanica and the bioceramic scaffold loaded with bone marrow mesenchymal stem cells are assembled into a composite scaffold, realizing the in-vitro osteogenic differentiation of bone marrow mesenchymal stem cells. The assembly of the pre-myogenic hydrogel scaffold and the bioceramic scaffold promotes the repair of muscle-skeleton complex tissue damage in vivo.

[0030] The assembled structure provided by the application can keep stable during the culture process, the cell survival state in the scaffold is good, and the cross-species scaffold has good biocompatibility. Therefore, the living plant cell-animal cell composite scaffold can be used as a multifunctional biomaterial to realize the living diatom photosynthesis and inorganic ion-induced differentiation in the cross-species structure, further domestic the surrounding tissue regeneration, and effectively solve the complex sports medical injury repair problem.

[0031] The application adopts: (1) structural bionics, the adopted bioceramic scaffold is combined with the hydrogel scaffold, which can effectively simulate the "soft tissue-hard tissue" surrounding structure of "muscles surrounding bones", and is closer to the real physiological state; the bioceramic scaffold provides the initial rigid mechanical support required by the composite structure, which is crucial for the integrity of the entire implant structure and the direction of guided tissue regeneration; (2) zoned culture, using the hydrogel scaffold or the bioceramic scaffold, selectively loading different cell components according to the real physiological structure, optimizing the cell culture microenvironment, and avoiding the interference problem caused by traditional scaffold cell mixed culture.

[0032] The two scaffolds not only maintain the independence of their respective microenvironments, but also need to achieve efficient biological integration at the interface. The performance of this synergistic effect depends on the control of key conditions and parameters during preparation, including: surface roughness and porosity of the bioceramic scaffold; viscosity and crosslinking of the hydrogel scaffold precursor solution; cell inoculation method and timing; interface zone biological functionalization design; scaffold thickness and permeability, etc.

[0033] Previous studies are mostly single muscle tissue regeneration or single bone defect repair, and few studies involve the overall recovery of the muscle-bone interface system; or the myogenic / osteogenic factors and cells are directly mixed in the same scaffold material, which lacks clear spatial separation and bionic gradient structure, and it is difficult to form an ordered tissue interface. The main technical challenges are: interface integration and stability, achieving stable and durable physical combination of the hydrogel scaffold and the bioceramic scaffold, preventing automatic delamination after culture or implantation; the hydrogel may exert stress on the ceramic scaffold during swelling, shrinking or degradation, leading to structural deformation or rupture; the swelling rate, degradation rate and mechanical properties of the two need to be matched; the wrapping structure may affect the material exchange (oxygen, nutrients, metabolic waste) in the internal ceramic scaffold area, especially when the hydrogel layer is thick or dense, which may cause the death of bone marrow mesenchymal stem cells in the core area due to hypoxia or nutrient deficiency, and the permeability and overall structure design of the hydrogel need to be optimized; the sterilization of the composite scaffold is more complex than that of a single material, the sterilization of the hydrogel scaffold needs to use a 0.22 μm filter to filter the methacrylated gelatin solution before the bio-ink is configured, and the sterilization of the bioceramic scaffold needs to use a high-pressure sterilization pot and put it into an oven for drying.

[0034] Hereinafter, a preparation method of the living plant cell-animal cell composite scaffold provided by the present application is exemplarily described. The preparation method can include the following steps: (1) mixing the functional plant cell component diatom and the mammalian cell component muscle stem cell with the high polymer matrix component hydrogel solution to obtain diatom-based pre-gel bio-ink and muscle stem cell-based pre-gel bio-ink, respectively, then pre-cooling and performing first 3D printing to obtain a cross-species symbiotic hydrogel scaffold with diatoms and muscle stem cells; (2) mixing the bioceramic scaffold matrix component with light-cured resin to obtain a precursor slurry, then performing second 3D printing, and obtaining a bioceramic scaffold matrix through solidification and sintering; then inoculating the mammalian cell component bone marrow mesenchymal stem cells on the bioceramic scaffold matrix to obtain the bioceramic scaffold; (3) assembling the hydrogel scaffold and the bioceramic scaffold to simulate a "muscle-skeleton" structure to obtain the living plant cell-animal cell composite scaffold.

[0035] In some embodiments, in step (1), the mammalian cell component muscle stem cell can be used in the form of a living cell suspension; the specific process can include: collecting the muscle stem cells with a confluence of 70-90% after digestion and centrifugation, and resuspending in medium to prepare a cell suspension.

[0036] In some embodiments, in step (1), the preparation method of the high polymer matrix component hydrogel solution can include: heating to dissolve the methacrylated gelatin in water at 55-60°C in a water bath under light shielding, adding a light initiator lithium phenyl-2,4,6-trimethylbenzoyl phosphinate, then filtering with a 0.22-0.45 μm filter in an ultra-clean bench to obtain a methacrylated gelatin hydrogel solution.

[0037] In some embodiments, in step (1), in the diatom-based pre-gel bio-ink, the concentration of the functional plant cell component diatom cell can be (0.1-10) × 10 6 / mL, preferably 1 × 10 6 / mL; and in the muscle stem cell-based pre-gel bio-ink, the concentration of the mammalian cell component muscle stem cell can be (2-4) × 10 6 / mL.

[0038] In some embodiments, in step (1), the pre-cooling is performed under light shielding, at a temperature of 0-8°C for 10-20 minutes, preferably sealed and stored in a 4°C refrigerator for 15 minutes to form a gel state.

[0039] In some embodiments, in step (1), the process of the first 3D printing can include: first depositing the muscle stem cell-based pre-gel bio-ink to a height of 1-6 mm or 5-30 layers, preferably 3 mm or 15 layers, and then depositing the diatom-based pre-gel bio-ink to a height of 1-6 mm or 5-30 layers, preferably 3 mm or 15 layers, and after the accumulation of layers is completed, blue light irradiation treatment is performed to form a two-section layered cross-species scaffold.

[0040] In some embodiments, in step (2), the photocurable resin can include W200 water-washed photosensitive resin; preferably, the preparation process of the precursor slurry can include: mixing 20-45 g of β-tricalcium phosphate powder with 25-55 g of photocurable resin, placing it in a ball mill for 1-3 h, filtering to obtain it, and storing it in the dark for later use.

[0041] In some embodiments, in step (2), the second 3D printing can be designed by Solidworks 2019 software to design the 3D structure of the scaffold, and the precursor slurry is crosslinked based on digital light processing 3D printing technology; wherein the parameters of the second 3D printing can include: 5-8 s of exposure time per 40-70 μm layer thickness, preferably 6 s of exposure time per 50 μm layer thickness.

[0042] If the exposure time is too short, the resin layer hardness is insufficient, the details are not shaped, and it is easy to break. If the exposure time is appropriate, it can ensure that the printing process is stable, the shape fidelity is high, the surface is relatively smooth, and the features are clear.

[0043] In some embodiments, in step (2), after the second 3D printing program is completed, the printer sample table can be removed, and the scaffold can be washed with ultrapure water 3-5 times, each time for 5-10 min, and washed with anhydrous ethanol 2-3 times.

[0044] In some embodiments, in step (2), the curing method can include ultraviolet light irradiation.

[0045] In some embodiments, in step (2), the sintering temperature can be 1000-1200°C, and the sintering time can be 2-4 h, preferably sintering at 1150°C for 3 h.

[0046] In some embodiments, in step (2), the mammalian cell component bone marrow mesenchymal stem cells can be used in the form of a live cell suspension; the specific process can include: bone marrow mesenchymal stem cells with a confluence of 70%-90% are collected after digestion and centrifugation, and are resuspended in MEM-α medium to form a cell suspension.

[0047] In some embodiments, in step (2), the seeding load concentration of the mammalian cell component bone marrow mesenchymal stem cells can be (0.5-2) x 105 1 x 10 5 1 x 10

[0048] In addition, the culture condition of the living plant cell-animal cell composite scaffold provided by the present application is as follows: 1 mL of mixed DMEM / ERDM medium (ratio: 4:1, 3:1, 2:1) is used to culture muscle stem cells at 37°C and 5% CO2, the culture condition of the mammalian component is screened, then the functional component Chaetoceros is cultured under the condition to ensure the survival of Chaetoceros, and the symbiotic culture condition is determined; the mixture of mammalian cell culture medium DMEM and bone marrow mesenchymal stem cell culture medium MEM-α, and the ratio of mammalian cell culture medium MEM-α to DMEM is 1:1. When the ratio of mammalian cell culture medium DMEM to algal culture medium ERDM is kept at 4:1, 3:1 and 2:1, Chaetoceros can survive normally. When the ratio of mammalian cell culture medium DMEM to algal culture medium ERDM is kept at 3:1, the expression degree of key factors of muscle differentiation and bone induction factors generated by muscle is higher, which is beneficial to the differentiation of muscle stem cells.

[0049] The living plant cell-animal cell composite scaffold provided by the present application can be applied to the field of muscle-skeletal injury repair, and the composite scaffold effectively promotes the repair and regeneration of muscle-skeletal injury in vivo and has good biocompatibility and biosafety. Benefiting from the release of silicon ions and oxygen production by photosynthesis of functional Chaetoceros in the scaffold, muscle stem cells are differentiated in the direction of myoblasts; in addition, the composite scaffold first domesticates the muscle-derived scaffold to the direction of pre-myoblasts by using the Chaetoceros scaffold, then removes the Chaetoceros scaffold, and co-cultures the pre-myoblast scaffold with the β-tricalcium phosphate ceramic scaffold loaded with bone marrow mesenchymal stem cells, so as to domesticate the bone marrow mesenchymal stem cells to the direction of osteogenesis, and the pre-myoblast hydrogel scaffold domesticated by Chaetoceros can promote the bone marrow mesenchymal stem cells on the ceramic scaffold to differentiate in the direction of osteogenesis.

[0050] In the volume muscle loss-segmental radial defect model, muscle stem cell scaffold myogenic differentiation can be domesticated by Chaetoceros in advance, then Chaetoceros is removed, and a pre-myoblast hydrogel scaffold-ceramic scaffold domestication system is assembled to promote the repair of muscle-bone injury. By establishing a volume muscle loss-segmental radial defect model of New Zealand white rabbits, blank group (Blank), pure hydrogel scaffold-β-tricalcium phosphate scaffold group (GT), muscle stem cell scaffold-β-tricalcium phosphate scaffold group (MuT) and pre-myoblast scaffold-β-tricalcium phosphate scaffold group (PMuT) are set. After the cross-species scaffold is implanted at the operation site, the repair function of the scaffold is verified by gait analysis and Micro-CT test. The introduction of muscle-derived cells in the scaffold, together with endogenous bone marrow mesenchymal stem cells in the injury site, is beneficial to the efficient and high-quality repair and regeneration of muscle-bone.

[0051] The following examples are further illustrated in detail to explain the present application. It should also be understood that the following examples are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present application are within the scope of protection of the present application. The specific process parameters in the following examples are only one example in the appropriate range, i.e. those skilled in the art can make appropriate selection within the range through the description herein, and are not limited to the specific values in the following examples. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art. If not specifically indicated, the functional components of live Chattonella come from the National Water Organism Germplasm Repository Freshwater Algae Seed Bank; β-tricalcium phosphate comes from Kunshan Huaxiao Science and Technology New Material Co., Ltd.

[0052] Example 1

[0053] The preparation method of the living plant cell-animal cell composite scaffold provided in the present embodiment comprises the following steps: (1) Dissolve 0.05 g of initiator lithium phenyl-2,4,6-trimethylbenzoyl phosphinate in 20 mL of ultrapure water, add 1.2 g of porous foam, heat to 60°C in a water bath in the dark until the methylacrylated gelatin is completely dissolved, filter in an ultra-clean workbench with a 0.22 μm filter to obtain a 6% (w / v) methylacrylated gelatin hydrogel solution as a printing precursor; disperse Chattonella in the methylacrylated gelatin solution, the printing density of Chattonella is 10 6 individuals / mL as a diatom-based pre-gel bio-ink; disperse muscle stem cells in the methylacrylated gelatin solution, select the printing density of muscle stem cells according to the results of symbiotic culture, as a stem cell-based pre-gel bio-ink; according to the specified procedure of first depositing muscle stem cell precursor X15 layers, then depositing Chattonella precursor Y15 layers, layer-by-layer accumulation is carried out to form a cross-species symbiotic hydrogel scaffold with diatoms and muscle stem cells; (2) Mix 45 g of β-tricalcium phosphate powder with 55 g of photocurable resin, place in a ball mill for 3 h, filter to obtain a uniform precursor slurry, and store in the dark for later use; design the 3D structure of the β-tricalcium phosphate scaffold with the aid of Solidworks 2019 software; crosslink the precursor slurry based on digital light processing 3D printing technology, adjust the printing parameters, and set the exposure time to 6 s per 50 μm layer thickness; after the printing program is completed, remove the printer sample table, wash the scaffold with ultrapure water for 3 times, each time for 5 min, and wash with anhydrous ethanol for 2 times; irradiate with ultraviolet light to solidify the scaffold, sinter at 1150°C for 3 h to obtain a β-tricalcium phosphate bioceramic scaffold matrix; then, inoculate mammalian cell component bone marrow mesenchymal stem cells on the bioceramic scaffold matrix to obtain the bioceramic scaffold; (3) simulate the muscle-bone structure, assemble the hydrogel scaffold with the bioceramic scaffold to obtain the living plant cell-animal cell composite scaffold.

[0054] Figure 1 For the physical and chemical properties of the β-tricalcium phosphate bioceramic scaffold based on digital light processing 3D printing in Example 1: (a) optical photo of the β-tricalcium phosphate bioceramic scaffold; (b) SEM image of the β-tricalcium phosphate bioceramic scaffold; (c) XRD pattern of the β-tricalcium phosphate bioceramic scaffold; (d) compressive strength-displacement curve of the β-tricalcium phosphate bioceramic scaffold; (e) theoretical value and measured value of the porosity of the β-tricalcium phosphate bioceramic scaffold. Figure 1 (a) shows that the designed and printed β-tricalcium phosphate bioceramic scaffold has a radius of 4 mm and a height of 6 mm; Figure 1 (b) shows that the struts are concentric hollow tubular structures that are completely penetrated in the center, with an outer diameter of 1 mm and an inner diameter of 600 μm, and are stacked in a 90° cross; Figure 1 (c) shows that the scaffold is a high-purity β-TCP hexagonal system; Figure 1 (d) shows that the compressive strength of the scaffold is about 1.7 MPa; Figure 1 (e) shows that the porosity of the scaffold is 73%, which is not significantly different from the theoretical value.

[0055] Figure 2 For the basic characteristics of Chaetoceros and scaffold assembly in Example 1: (a) laboratory culture of Chaetoceros; (b) SEM image of Chaetoceros; (c) UV-vis spectrum of Chaetoceros; (d) photo of Chaetoceros scaffold; (e) change curve of dissolved oxygen content of Chaetoceros-containing hydrogel scaffold under 5000 lx light-emitting diode irradiation at 25℃ or 37℃; (f) cumulative release curve of silicon ions of Chaetoceros-containing hydrogel scaffold at 37℃; (g) photo of nested assembly of methacrylated hydrogel scaffold and β-tricalcium phosphate bioceramic scaffold. Figure 2 (a) laboratory culture of Chaetoceros was achieved, and under the conditions of constant temperature at 25℃ and light intensity of 2500 lx, Chaetoceros grew well under the light-dark rhythm of 12 h light / 12 h dark and could be precisely replicated on a large scale; Figure 2 (b) shows that Chaetoceros is a unicellular microalgae, with an algal body length of about 5 μm and a hollow bristle at the top; Figure 2 (c) shows that the chloroplast of Chaetoceros mainly contains chlorophyll and carotenoids; Figure 2 (d) is an extrusion-printed Chaetoceros scaffold; Figure 2 (e) determination, 1×10 6 mg / L of Chaetoceros scaffold under 5000 lx light intensity for 70 minutes at 37℃ produced a change value of dissolved oxygen content of 2.55±0.31 mg / L;Figure 2 (f) indicating that the g. oceanica scaffold can degrade continuously release silicon ions; Figure 2 (g) indicating that the bio-ceramic scaffold and the hydrogel scaffold can be freely assembled. The above results show that the composite scaffold assembled by the bio-ceramic scaffold and the hydrogel scaffold has excellent basic properties, can degrade to release silicon ions and produce oxygen through photosynthesis, and the scaffold provides favorable conditions for inducing cell adhesion and differentiation.

[0056] Determination of the living plant cell-animal cell cross-species hydrogel scaffold symbiotic culture conditions

[0057] Considering the physiological environment of mammals, the Transwell method was used to explore the muscle stem cell and g. oceanica cell ratio (2:1, 3:1 or 4:1) and symbiotic culture conditions; the hydrogel scaffold containing only muscle stem cells (xMuG), the living plant cell-animal cell cross-species scaffold (xMuD) placed in a dark environment or the living plant cell-animal cell cross-species scaffold (xMuL) (x=2, 3 or 4) subjected to rhythmic light were maintained at the same ratio of medium and cells. Calcein-AM / PI staining was used to evaluate the muscle stem cell viability in the living plant cell-animal cell cross-species scaffold. Further optimization of the cell ratio and culture cycle of the living plant cell-animal cell cross-species biphasic scaffold, the symbiotic scaffold was cultured in a well plate for 7 days and fixed with 4% paraformaldehyde. Total ribonucleic acid (RNA) was isolated from mammalian cells in each bioprinted scaffold using RNAiso, chloroform and isopropanol, reverse transcribed using PrimeScriptTM, and the expression of muscle differentiation-related genes (MYOD and PAX7) and osteogenesis-related genes secreted by muscle (FAM5C and OGN) was detected by reverse transcription polymerase chain reaction (RT-PCR).

[0058] Figure 3 For the biocompatibility of the living plant cell-animal cell cross-species hydrogel scaffold in Example 1: muscle stem cells and g. oceanica cells were co-cultured for 7 days at a ratio of (a) 2:1, (b) 3:1 and (c) 4:1, and the fluorescence images of muscle stem cells in the scaffold were stained with calcein AM / PI. As can be seen from Figure 3 , the living plant cell-animal cell cross-species hydrogel scaffold showed green fluorescence and maintained good growth activity in the 7-day light / dark cycle, and the cross-species biphasic scaffold had good biocompatibility; the fluorescence intensity of the 3MuL group increased significantly after 7 days of culture, and the number of cells showed an increasing trend; the fluorescence signal of the 4MuL group was the strongest after 4 days of culture, but it decreased on the 7th day, suggesting that the living space of the scaffold was relatively limited, and cell shedding occurred after excessive cell proliferation, which may also be related to the accumulation of metabolic waste.

[0059] Figure 4Gene expression levels of the living plant cell-animal cell cross-species hydrogel scaffold in Example 1: After the living plant cell-animal cell cross-species hydrogel scaffold was cultured for 7 days, the expression of (a) muscle regeneration key factors (MYOD) and stem cell marker genes (PAX7) produced by muscle stem cells, and (b) bone induction factors (OGN and FAM5C) produced by muscle stem cells. Figure 4 (a) shows that the 3MuL (7 days) group and the 4MuL (4 days) group have similar expression of muscle growth and regeneration key factors (MYOD); Figure 4 (b) shows that the 3MuL (7 days) group has significantly higher expression of bone induction factors such as OGN, FAM5C, etc. produced by muscle than the 4MuL (4 days). Based on the above results, the living plant cell-animal cell cross-species hydrogel scaffold is well co-cultured, and the ratio of muscle stem cells to chaetophora cells is preferably 3:1, and the culture is carried out at 37°C for 7 days.

[0060] When determining the co-culture conditions, the muscle stem cell scaffold is pre-myogenic by the chaetophora scaffold, and the ratio of muscle stem cells to chaetophora and whether to apply light all affect the growth of the muscle stem cell scaffold; when the cell ratio is 3:1 and the co-culture is carried out for 7 days with rhythmic light, the myogenic differentiation effect of the muscle stem cell scaffold is the best, and the effects of other groups are poor. The Control group is a single muscle stem cell scaffold without chaetophora, and the survival is the worst. Figure 3 and Figure 4 ).

[0061] Myogenic and osteogenic differentiation of the living plant cell-animal cell composite scaffold

[0062] In order to better explore the relationship between chaetophora, muscle and bone in the cross-species scaffold, according to the results of the previous experiments and early literature research, a possible action route is assumed, that is, using a Transwell chamber, the MuG scaffold is pre-conditioned by the chaetophora scaffold, which promotes the myogenic differentiation of the muscle stem cells in the MuG scaffold, and the groups are set as pure muscle stem cell group (Control), chaetophora scaffold and MuG scaffold coexistence dark group (MuD) and chaetophora scaffold and MuG scaffold coexistence light group (MuL). Subsequently, the chaetophora scaffold is removed, and the pure methacrylated gelatin scaffold, muscle stem cell scaffold and differentiated pre-myogenic scaffold are assembled and cultured with the β-tricalcium phosphate scaffold loaded with BMSC, respectively, and the groups are named as Control, MuB and PMuB in turn. The F-actin cytoskeleton morphology of muscle stem cells and bone marrow mesenchymal stem cells in the cross-species scaffold is labeled by Alex Fluor 647 conjugated phalloidin, and the expression of myogenic related proteins (MHC and SCA) and osteogenic related proteins (OPN and BMP2) is characterized by immunofluorescence staining. Fluorescence images are taken by laser confocal microscope (CLSM).

[0063] Figure 5 Fig. 2 shows the images of immunofluorescence staining of F-actin in the muscle stem cells in the scaffolds after 7 days of culture (a) and the images of immunofluorescence staining of myogenic-related proteins (MHC and SCA) in the muscle stem cells in the scaffolds after 7 days of culture (b). Figure 5 (a) shows that the muscle stem cells in the BMuL group exhibit better elongation and spreading after 7 days, the muscle stem cells are stretched, extended from the hydrogel column to the surface, and spread to form a uniform and dense F-actin network; Figure 5 (b) shows that the expression of MHC and SCA in the MuG scaffold is promoted by the Chaetopterus scaffold, and the protein expression level in the MuL group is increased after the application of rhythmic light, proving that the cross-species scaffold accelerates myogenic differentiation by releasing silicon ions and oxygen generated by photosynthesis of pigments from Chaetopterus.

[0064] Figure 6 Fig. 4 shows the images of immunofluorescence staining of F-actin in the muscle stem cells in the scaffolds after 7 days of culture (a) and the images of immunofluorescence staining of osteogenic-related proteins (OPN and BMP2) in the bone marrow mesenchymal stem cells in the scaffolds after 7 days of culture (b). Figure 6 (a) shows that the cells in each group can extend and grow along the β-tricalcium phosphate pillars and spread on the surface to form a protein network, and the F-actin network in the BMuL group is more dense and orderly, and the stress fiber arrangement characteristic of mature muscle cells can be seen; Figure 6 (b) shows that the OPN and BMP2 in the MuB group exhibit stronger fluorescence, and the fluorescence level in the PMuB group is higher than that in the MuB group, proving that the pre-myogenic scaffold induced by cross-species domestication effectively promotes the osteogenic differentiation process. The above results show that the living plant cell-animal cell composite scaffold effectively domesticates muscle stem cells for myogenic differentiation, and the pre-differentiated muscle scaffold can promote osteogenic differentiation.

[0065] In the myogenic-osteogenic differentiation experiment of the composite scaffold in vitro, the pre-myogenic scaffold induces the osteogenic differentiation of the bone marrow mesenchymal stem cell scaffold, and the Control group is a pure methacrylated gelatin scaffold combined with a bioceramic scaffold loaded with bone marrow mesenchymal stem cells, which has poor osteogenic differentiation effect and is significantly different from other groups Figure 6 .

[0066] Repair effect of composite scaffold on rabbit volume muscle loss-segmental radial defect

[0067] New Zealand white rabbits (male, 6 months old) were selected to establish a rabbit volumetric muscle loss-segmental radial defect model. They were randomly divided into 4 groups: 12 hours before surgery, they were prohibited from feeding and drinking water. The rabbits were anesthetized and fixed on the side, a 2 cm longitudinal incision was made in the middle of the forearm, the skin and fascia were successively incised, and the radial bone was exposed by bluntly separating the forearm muscles. A 10 mm segment of radial bone was transversely cut off using a swing saw. At the same time, a bundle of muscle tissue 10 mm long and 30% thick was cut from the forearm muscle around the radial bone. Before the operation, GelMA scaffolds without any cell components, muscle stem cell scaffolds MuG, and differentiated muscle scaffolds after domestication of Chaetoceros (PMuG, P means pre-differentiation) were respectively bioprinted and assembled with β-tricalcium phosphate scaffolds to simulate the physiological model of muscle-bone, which were recorded as GT, MuT, and PMuT groups. The scaffolds were implanted at the defect site. The treatment period was 12 weeks. At 4 and 12 weeks after the operation, the dynamic spatial parameters (left and right step length, stride length, and stride width) and paw parameters (toe width and paw length) were collected by using a custom-made acrylic walkway (length x width x height = 300 x 40 x 35 cm 3 ), and gait analysis was performed. At 4 and 12 weeks after the operation, the rats were sacrificed, and the bone defects were reconstructed using Micro-CT.

[0068] Figure 7 The surgical procedure for the composite scaffold used in the rabbit volumetric muscle loss-segmental radial defect in Example 1.

[0069] Figure 8 The repair effect of the composite scaffold used in the rabbit volumetric muscle loss-segmental radial defect in Example 1 was analyzed by gait analysis: (a) is a representative paw print at 4 and 12 weeks after the operation; (b) is the asymmetry index of paw parameters and spatial parameters at 4 and 12 weeks after the operation, including paw length, toe width, step length, stride length, and step width. Figure 8 As can be seen in (b), at 4 weeks after the operation, although there were obvious differences in inter-toe distance and stride length parameters in each group, the gait on the surgical side was abnormal; at 12 weeks after the operation, the GT group only partially recovered, the MuT group and the PMuT group increased in step length and step width, and the movement function was partially restored, and the AI index of the PMuT group continuously improved and almost recovered to the normal level.

[0070] Figure 9 The repair effect of the composite scaffold used in the rabbit volumetric muscle loss-segmental radial defect in Example 1 was evaluated by histology: (a) is the Micro-CT image and bone volume fraction (BV / TV) value of the muscle-radial sample at 4 weeks after the operation; (b) is the Micro-CT image and bone volume fraction (BV / TV) value of the muscle-bone sample at 12 weeks after the operation. Figure 9 As shown in (a), at 4 weeks after the operation, the defects in each group were slightly recovered, and there was little new bone tissue, and the BV / TV value of the PMuT group was about 13%; Figure 9(b) It is shown that the bone regeneration ability of the blank group is still limited at 12 weeks after operation, the MuT group has some recovery but is still not ideal, and the PMuT group can observe obvious new tissue growth into the cross-species scaffold, and the BV / TV value is about 32%, which proves that the PMuT has significant bone repair ability. The above results show that the muscle-bone gradually rebuilds over time, and the composite scaffold has excellent bone integration ability.

[0071] In the experiment of repairing rabbit volume muscle loss in vivo by using the composite scaffold, the GT group is a pure methylacrylated gelatin scaffold combined with a pure β-tricalcium phosphate bioceramic scaffold, and the recovery of motor function is poor Figure 8 ), and the bone damage repair effect is poor, and the amount of new bone is much less than that of other groups Figure 9 ).

[0072] The above series of experiments verify the good effect of the living plant cell-animal cell composite scaffold for tissue repair and regeneration.

[0073] Although the content of the present application has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as limiting the present application. After reading the above content, various modifications and alternatives of the present application will be apparent to those skilled in the art. Therefore, the protection scope of the present application should be defined by the appended claims.

Claims

1. A living plant cell-animal cell composite scaffold, characterized in that, The living plant cell-animal cell composite scaffold comprises a 3D-printed hydrogel scaffold assembled to mimic the "muscle-skeleton" structure, serving as a muscle repair carrier, and a 3D-printed bioceramic scaffold serving as a bone repair carrier; wherein... The hydrogel scaffold comprises: a polymer matrix component, and functional plant cell components (diatom cells) and mammalian cell components (muscle stem cells) distributed in the polymer matrix component; The bioceramic scaffold comprises: a bioceramic scaffold matrix component, and bone marrow mesenchymal stem cells, a mammalian cell component, seeded onto the bioceramic scaffold matrix component.

2. The living plant cell-animal cell composite scaffold according to claim 1, characterized in that, The polymer matrix component includes methacrylamide gelatin; the functional plant cell component, diatom cells, includes Chaetoceros cells; and the bioceramic matrix component includes β-tricalcium phosphate.

3. The living plant cell-animal cell composite scaffold according to claim 1 or 2, characterized in that, In the hydrogel scaffold, the mass concentration of the polymer matrix component can be 4-8%, preferably 6%; the concentration of the functional plant cell component, diatom cells, is (0.1-10)×10⁻⁶. 6 Cells / mL, preferably 1×10⁻⁶ 6 The concentration of the mammalian cell component, muscle stem cells, was (2-4)×10⁻⁶ / mL. 6 cells / mL; In the bioceramic scaffold, the seeding concentration of the mammalian cell component, bone marrow mesenchymal stem cells, is (0.5-2)×10⁻⁶. 5 Cells / mL, preferably 1×10⁻⁶ 5 per mL.

4. A method for preparing a living plant cell-animal cell composite scaffold according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: (1) The functional plant cell component diatom and the mammalian cell component muscle stem cell were respectively mixed with the polymer matrix component hydrogel solution to obtain diatom-based pregel bio-ink and muscle stem cell-based pregel bio-ink. After pre-cooling, the first 3D printing was performed to obtain a cross-species symbiotic hydrogel scaffold with both diatoms and muscle stem cells. (2) The bioceramic scaffold matrix components are mixed with photocurable resin to obtain a precursor slurry, which is then subjected to a second 3D printing process. After curing and sintering, the bioceramic scaffold matrix is ​​obtained. Next, mammalian cell components, bone marrow mesenchymal stem cells, are seeded and loaded onto the bioceramic scaffold matrix to obtain the bioceramic scaffold. (3) The hydrogel scaffold and the bioceramic scaffold are assembled to simulate the "muscle-skeleton" structure to obtain the living plant cell-animal cell composite scaffold.

5. The preparation method according to claim 4, characterized in that, In step (1), the preparation method of the polymer matrix component hydrogel solution includes: heating in a water bath at 55-60℃ in the dark until the methacrylamide gelatin dissolves in water, adding the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, and filtering to obtain the solution.

6. The preparation method according to claim 4 or 5, characterized in that, In step (1), the first 3D printing process includes: first depositing 1-6 mm or 5-30 layers of muscle stem cell-based pregel bio-ink, then depositing 1-6 mm or 5-30 layers of diatom-based pregel bio-ink, and after layer-by-layer accumulation, performing blue light irradiation treatment to form a two-stage layered cross-species scaffold.

7. The preparation method according to any one of claims 4-6, characterized in that, In step (2), the preparation process of the precursor slurry includes: mixing 20-45g of β-tricalcium phosphate powder with 25-55g of photocurable resin, placing it in a ball mill for 1-3 hours, and filtering to obtain the slurry.

8. The preparation method according to any one of claims 4-7, characterized in that, In step (2), the parameters for the second 3D printing include: an exposure time of 5-8s for every 40-70μm layer thickness, preferably 6s for every 50μm layer thickness.

9. The preparation method according to any one of claims 4-8, characterized in that, In step (2), the sintering temperature is 1000-1200℃ and the sintering time is 2-4h, preferably sintering at 1150℃ for 3h.

10. The application of any one of claims 1-3 in the field of muscle-bone injury repair using a living plant cell-animal cell composite scaffold.

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