Cell-laden microfibers, preparation methods, root canal implants and applications

Abstract

The present invention provides cell-loaded microfibers, preparation methods, root canal implants and applications. The embodiments of the present invention determine a suitable concentration of GelMA hydrogel, which can promote the survival and proliferation of DPSCs and human umbilical vein endothelial cells (HUVECs), and has good biomechanical properties to produce microfibers. Two GelMA microfibers of DPSCs and HUVECs were prepared by a silicon tube-based coagulant-free bath method. The results of live / dead fluorescence staining and Ki-67 immunofluorescence staining showed that these two cells maintained a high survival rate and proliferation ability in GelMA microfibers. Immunofluorescence staining confirmed that DPSCs were fully diffused in the microfibers, and CD90 and laminin were highly expressed. In the embodiments of the present invention, the nude mouse ectopic dental pulp regeneration experiment was used to verify the regeneration of DPSC-microfiber and HUVEC-microfiber mixed aggregates in vivo. Compared with the traditional mixture of DPSCs and HUVECs in the GelMA hydrogel block, the cell-loaded microfiber aggregation produced more pulp-like tissue, blood vessels and odontoblast-like cells positively expressing DMP-1 and DSPP.

Description

Technical Field

[0001] The present invention relates to the field of basic biomedical research, and in particular to a cell-laden microfiber, a preparation method, a root canal implant and applications. Background Art

[0002] Teeth have a unique structure, including internal soft tissue (pulp) and external hard tissue (enamel and dentin). The pulp contains connective tissue, blood vessels, nerves and lymphatic vessels, and plays an important role in regulating tooth sensation and promoting dentin regeneration. However, deep caries, trauma and microbial infection may lead to irreversible pulp inflammation and necrosis. Currently, root canal therapy is the main treatment strategy for these diseases, which involves removing the pulp with inorganic fillings, blocking the root canal, resulting in termination of root development, and limited survival time of the treated teeth. Therefore, there is an urgent need to regenerate the important dental pulp to restore its function.

[0003] Currently, a new treatment model for pulp necrosis is dental pulp stem cell (DPSCs) transplantation to regenerate lost pulp. DPSCs transplantation has been used in clinical trials for pulp regeneration. However, DPSC transplantation is only applicable to young permanent teeth, which is due to the wide and incomplete root canal openings of young permanent teeth, which is conducive to sufficient exchange of nutrients and gases to support the survival and function of transplanted stem cells. The apical foramen of mature permanent teeth is small, which is not conducive to material exchange and vascularization, and DPSCs transplantation cannot be used. Therefore, vascularization of pulp tissue regeneration is very important. Conventional hydrogel blocks or stem cell aggregates have poor permeability, resulting in a negative microenvironment lacking nutrients and oxygen for the regeneration of these cell lines. In mature teeth with hypoxia and malnutrition in the narrow root canal microenvironment, it is still a huge challenge to restore the vitality of the dental pulp with transplanted DPSCs.

[0004] Therefore, a new strategy for dental pulp regeneration is urgently needed. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned background technology, the present invention provides a cell-laden microfiber, a preparation method, a root canal implant and application.

[0006] In a first aspect, the present invention provides a cell-loaded microfiber, wherein the cell-loaded microfiber is a methacrylated gelatin microfiber loaded with dental pulp stem cells or human umbilical vein endothelial cells; the cell-loaded microfiber is used to mimic the structure of dental pulp.

[0007] Optionally, the cell content in the cell-laden microfiber is 0.5*10 7 cell / ml-2*10 7 cell / ml.

[0008] Optionally, the concentration of the methacrylated gelatin solution in the cell-laden microfibers is 50-100 mg / ml.

[0009] Optionally, the diameter of the cell-laden microfibers is 300-500 μm.

[0010] In a second aspect, the present invention provides a method for preparing cell-laden microfibers, the method comprising:

[0011] Prepare dental pulp stem cells or human umbilical vein endothelial cells;

[0012] The dental pulp stem cells or human umbilical vein endothelial cells are respectively mixed with the methacrylated gelatin solution to obtain a matrix solution;

[0013] The matrix solution is injected into a transparent silicone tube, and then the transparent silicone tube is exposed to ultraviolet light for 30 seconds; the matrix solution is gradually cross-linked in the transparent silicone tube to form microfibers;

[0014] The microfiber is pushed out of the transparent silicone tube to obtain the cell-laden microfiber.

[0015] In a third aspect, the present invention provides a root canal implant, which is composed of two types of cell-laden microfibers, including methacrylated gelatin microfibers carrying dental pulp stem cells and methacrylated gelatin microfibers carrying human umbilical vein endothelial cells.

[0016] Optionally, in the root canal implant, the ratio of the methacrylated gelatin microfibers loaded with dental pulp stem cells to the methacrylated gelatin microfibers loaded with human umbilical vein endothelial cells is 2:1.

[0017] Optionally, the methacrylated gelatin microfibers loaded with dental pulp stem cells and the methacrylated gelatin microfibers loaded with human umbilical vein endothelial cells included in the root canal implant are randomly entangled to form microfiber aggregates.

[0018] In a fourth aspect, the present invention provides an application of a root canal implant, wherein the root canal implant is used to mimic the structure of a dental pulp and is applied to the construction of a repair module for a necrotic dental pulp.

[0019] Optionally, the root canal implant improves pulp blood supply reconstruction through a microfiber structure, promotes pulp regeneration through implantation of dental pulp stem cells, and promotes blood vessel formation in regenerated pulp through implantation of human umbilical vein endothelial cells.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The cell-laden microfibers proposed in the embodiments of the present invention can promote the survival and proliferation of DPSCs and human umbilical vein endothelial cells (HUVECs), and these two cells maintain a high survival rate and proliferation ability in GelMA microfibers. Immunofluorescence staining confirmed that DPSCs were fully diffused in the microfibers, and CD90 and laminin were highly expressed. HUVECs positively expressed CD31 and VE-cad in the microfibers and could migrate well in the GelMA hydrogel. In vitro permeation experiments confirmed that microfiber aggregates were superior to GelMA hydrogel blocks in terms of liquid permeability. The subcutaneous ectopic dental pulp regeneration experiment in nude mice confirmed that the root canal implants obtained using cell-laden microfibers can quickly form regenerated dental pulp in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a method flow chart of a method for preparing cell-laden microfibers provided in an embodiment of the present invention;

[0023] Figure 2 is a schematic diagram of the application process of the implant provided by an embodiment of the present invention;

[0024] Figure 3 1 is a graph showing the characterization results of three concentrations of GelMA hydrogels prepared in the examples of the present invention;

[0025] Figure 4 is a graph showing the viability and proliferation ability of cells loaded in microfibers prepared in an embodiment of the present invention;

[0026] Figure 5 This is a result diagram of cell staining fluorescence image of microfibers prepared in an embodiment of the present invention;

[0027] Figure 6 is a graph showing the permeability evaluation results of the GelMA microfiber aggregate (MA) prepared in an embodiment of the present invention;

[0028] Figure 7 is a diagram showing the evaluation results of the new tissue of ectopic dental pulp regeneration in an embodiment of the present invention;

[0029] Figure 8 is a diagram showing the angiogenesis results in an ectopic dental pulp regeneration test in an embodiment of the present invention;

[0030] Fig. 9 1 is a diagram showing the evaluation results of the cell sources for regenerating dental pulp tissue and blood vessels in an embodiment of the present invention. DETAILED DESCRIPTION

[0031] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0032] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. The reagents and other instruments used without specifying the manufacturer are all conventional reagent products that can be purchased on the market.

[0033] Dental pulp stem cell transplantation to regenerate dental pulp is a new method for treating pulp necrosis after inflammatory injury. However, how to provide sufficient oxygen and nutrients in the narrow root canal to support the survival, self-renewal and differentiation of stem cells remains a huge challenge.

[0034] To accelerate angiogenesis in dental pulp, endothelial cells or microvascular fragments can be mixed with DPSCs before transplantation. Cell-bearing microfibers are considered to be a promising engineering structure for promoting angiogenesis and tissue regeneration. First, hydrogel microfibers can serve as an ideal template for microvascular engineering. Cell-bearing microfibers can be arranged in different forms to meet the needs of vascularization. Second, cell-bearing microfibers provide a unique platform and biocompatible microenvironment for cell adhesion, growth, proliferation, and differentiation. Cells in microfibers can aggregate and connect to each other for further tissue formation. Third, microfibers can be used as basic units to construct macroscopic buildings with personalized structures. Finally, the aggregation of microfibers forms a loose three-dimensional macrostructure with tiny gaps between fibers, providing channels for fluid and gas exchange between host cells and implanted cells. Taking advantage of these advantages, cell fibers have been applied to the regeneration of various tissues, including skeletal muscle, nerve, bone, smooth muscle, pancreatic islets, myocardium, and lung.

[0035] Gelatin methacrylate (GelMA) is a good biological scaffold material with good biocompatibility, mild cross-linking conditions and low immunogenicity, providing a good microenvironment for cell survival. In addition, the mechanical and biological properties of GelMA are adjustable. Based on the above advantages, GelMA has been used in many studies to prepare cell-loaded microfibers and achieved good results.

[0036] Therefore, the embodiment of the present invention believes that cell-loaded GelMA microfibers can promote dental pulp regeneration. Based on this, the embodiment of the present invention proposes to inject optimized GelMA loaded with dental pulp stem cells (Dental Pulp Stem Cells, DPSCs) or human umbilical vein endothelial cells: (Human Umbilical Vein Endothelial Cells, HUVEC) into a transparent silicone tube, and prepare cell-loaded microfibers by ultraviolet cross-linking. Two types of GelMA hydrogel microfibers loaded with HUVECs and DPSCs were prepared respectively, and combined into microfiber aggregates (MAs) in a ratio of 2:1 as root canal implants. After transplantation into the dental canal, MAs produced vascularized pulp-like tissue, and then, the embodiment of the present invention obtained a new dental pulp regeneration strategy.

[0037] In the examples of the present invention, the inventors further explored the new strategy of dental pulp regeneration based on cell-loaded microfibers proposed in the examples of the present invention. First, the examples of the present invention determined the GelMA hydrogel in a suitable proportion to promote the survival and proliferation of DPSCs and human umbilical vein endothelial cells (HUVECs), and to prepare microfibers with good biomechanical properties. In the examples of the present invention, two types of GelMA microfibers, DPSCs and HUVECs, were prepared by a silicon tube-based coagulant bath-free method. The results of live / dead and Ki-67 immunofluorescence staining showed that these two cells maintained a high survival rate and proliferation ability in GelMA microfibers. Immunofluorescence staining confirmed that DPSCs were fully diffused in the microfibers, and CD90 and laminin were highly expressed. HUVECs positively expressed CD31 and VE-cad in the microfibers and could migrate well in the GelMA hydrogel. In vitro permeation experiments confirmed that microfiber aggregates were superior to GelMA hydrogel blocks in terms of liquid permeability.

[0038] The present invention further uses a nude mouse ectopic dental pulp regeneration experiment to verify the in vivo regeneration of DPSC-microfiber and HUVEC-microfiber mixed aggregates. Compared with the traditional mixture of DPSCs and HUVECs in GelMA hydrogel blocks, the cell-loaded microfiber aggregates produced more pulp-like tissue, blood vessels, and odontoblast-like cells that positively expressed DMP-1 and DSPP.

[0039] Specifically, in a first aspect, an embodiment of the present invention provides a cell-loaded microfiber, wherein the cell-loaded microfiber is a methacrylated gelatin microfiber loaded with dental pulp stem cells or human umbilical vein endothelial cells; the cell-loaded microfiber is used to mimic the structure of dental pulp.

[0040] Specifically, the cell content in the cell-laden microfibers is 0.5*107cell / ml to 2*107cell / ml.

[0041] Specifically, the concentration of the methacrylated gelatin solution in the cell-laden microfibers is 50-100 mg / ml.

[0042] Specifically, the diameter of the cell-laden microfiber is 300-500 μm.

[0043] In the embodiment of the present invention, the diameter of the cell-laden microfibers is at the micron level, which has better mechanical properties than nanofibers. In addition, the diameter of the microfibers in the embodiment of the present invention is about 300-500 μm, and the preferred diameter is 400 μm, which is close to the diameter of microvessels and can better simulate the dental pulp structure.

[0044] The cell-laden microfibers provided in the embodiments of the present invention can be cultured or modified in vitro for more than one week, thereby expanding the application scope of the cell-laden microfibers and reducing the difficulty of clinical application.

[0045] In the embodiment of the present invention, the methacrylated gelatin microfibers can be prepared by photo-crosslinking for only 30 seconds.

[0046] In the embodiment of the present invention, the cell concentration in the cell-carrying microfiber is relatively high, reaching 1*10 7 cell / ml, which is more conducive to tissue formation.

[0047] In the embodiment of the present invention, DPSCs and HUVECs are respectively encapsulated in GelMA microfibers, which is beneficial to faster aggregation of HUVECs and formation of blood vessels.

[0048] In the embodiment of the present invention, the selected GelMA superfiber can provide a better living environment for HUVECs and improve their survival rate. Within one week, the survival rate of HUVECs in the GelMA superfiber remains at about 90%.

[0049] Specifically, in the second aspect, an embodiment of the present invention provides a method for preparing cell-laden microfibers, and the cell-laden microfibers described in the first aspect can be prepared by using this method.

[0050] like Figure 1 As shown, it shows a flow chart of a method for preparing a cell-laden microfiber provided by an embodiment of the present invention. Specifically, the method comprises the following steps:

[0051] S101: Prepare dental pulp stem cells or human umbilical vein endothelial cells.

[0052] S102: dental pulp stem cells or human umbilical vein endothelial cells are mixed with methacrylated gelatin solution to obtain a matrix solution.

[0053] S103: injecting the matrix solution into a transparent silicone tube, and then exposing the transparent silicone tube to ultraviolet light for 30 seconds; the matrix solution gradually cross-links in the transparent silicone tube to form microfibers.

[0054] S104: Pushing the microfiber out of the transparent silicone tube to obtain the cell-laden microfiber.

[0055] Specifically, in the third aspect, an embodiment of the present invention provides a root canal implant. The root canal implant is composed of two cell-carrying microfibers, and the cell-carrying microfibers include: methacrylated gelatin microfibers carrying dental pulp stem cells and methacrylated gelatin microfibers carrying human umbilical vein endothelial cells. The methacrylated gelatin microfibers carrying dental pulp stem cells and the methacrylated gelatin microfibers carrying human umbilical vein endothelial cells can be the cell-carrying microfibers provided in the first aspect of the embodiment of the present invention.

[0056] Specifically, in an embodiment of the present invention, in the root canal implant, the ratio of the methacrylated gelatin microfibers loaded with dental pulp stem cells to the methacrylated gelatin microfibers loaded with human umbilical vein endothelial cells is 2:1.

[0057] Specifically, in an embodiment of the present invention, the methacrylated gelatin microfibers loaded with dental pulp stem cells and the methacrylated gelatin microfibers loaded with human umbilical vein endothelial cells included in the root canal implant are randomly entangled to form microfiber aggregates.

[0058] Figure 2 The embodiment provides a schematic diagram of the application process of a root canal implant, such as Figure 2 As shown, in the embodiment of the present invention, the inner diameter of the transparent silicone tube is 400μm, so that microfibers with a diameter of 400μm can be prepared. In the embodiment of the present invention, the GelMA solution mixed with cells is injected into the transparent silicone tube, and then the tube is exposed to ultraviolet light for 30 seconds. The GelMA solution gradually cross-links in the transparent silicone tube to form microfibers. The microfibers are then pushed out with a needle of suitable diameter to obtain cell-carrying microfibers. After respectively obtaining microfibers carrying dental pulp stem cells and microfibers carrying human umbilical vein endothelial cells, the two are mixed, and the two are disorderly entangled to obtain a microfiber aggregate (MA). The microfiber aggregate is implanted into the recipient root cannula to achieve the regeneration of dental pulp tissue.

[0059] Specifically, in a fourth aspect, an embodiment of the present invention provides an application of a root canal implant, wherein the root canal implant is used to imitate the structure of a dental pulp and is applied to the construction of a repair module for a necrotic dental pulp.

[0060] Specifically, the root canal implant improves the blood supply reconstruction of the dental pulp through the microfiber structure, promotes the regeneration of the dental pulp through the implantation of dental pulp stem cells, and promotes the formation of blood vessels in the regenerated dental pulp through the implantation of human umbilical vein endothelial cells.

[0061] In order to enable those skilled in the art to better understand the present invention, the cell-laden microfibers, preparation methods, root canal implants and applications provided by the present invention are described below through specific embodiments.

[0062] Example 1: Preparation of cell-laden microfibers

[0063] 1. Exploration of GelMA concentration:

[0064] In the embodiment of the present invention, GelMA (GM-90) was purchased from Suzhou Intelligent Manufacturing Research Institute (Suzhou, China). The photoinitiator was LAP. According to the instructions, 0.5wt% of the photoinitiator was dissolved in a PBS solution at 45°C for 15 minutes. GelMA was dissolved in a solution containing the photoinitiator and then filtered with a 0.22mm filter for sterilization. Three concentrations of GelMA were prepared for the experiment, including 5% (w / v), 7.5% (w / w) and 10% (w / v), and the three concentrations of GelMA were injected into a 2mL EP tube and then cross-linked under 405nm (25mW / cm2) UV light for 30 seconds to prepare a cylindrical hydrogel block with a diameter of 1cm and a height of 6-8mm.

[0065] The three concentrations of GelMA were frozen in liquid nitrogen for 20 minutes and freeze-dried for 2 hours using a freeze dryer (SCIENTZ-18ND, China). In order to compare their internal pore sizes, the freeze-dried samples were cut into slices and coated with gold sputtering for scanning electron microscopy observation. The surface morphology of the freeze-dried microfibers was then observed using SEM. The pore sizes of three GelMA hydrogel composites (5%, 7.5% and 10%, w / v) were observed by SEM to select the optimal GelMA concentration.

[0066] DPSC was 1.5*10 6 The concentration of cells / mL was planted in these three concentrations of GelMA to observe the diffusion of cells. Specifically, DPSCs were implanted in GelMA solutions with the above three concentrations, and cell-loaded hydrogel blocks with a size of 10 μl were prepared by photocrosslinking. The prepared cell-loaded hydrogel blocks were cultured for 4 days according to the DPSCs culture method. The samples were fixed with 4% paraformaldehyde on the 1st and 4th days of culture, and then stained with DAPI (blue) and F-actin (red).

[0067] Figure 3The characterization result diagram of three concentrations of GelMA hydrogel prepared in the embodiment of the present invention is shown. Part A shows SEM images of three freeze-dried GelMA hydrogels (scale bar: 50 μm). Part B shows a quantitative analysis of the pore size of three GelMA hydrogels. Part C shows immunofluorescence images of DPSCs diffused in GelMA hydrogels on the 1st and 4th days of culture (scale bar, 50 μm), stained with DAPI (blue) and F-actin (red) after culture. Part D shows a quantitative analysis of the aspect ratio of DPSC cells in GelMA hydrogels.

[0068] Quantitative analysis of SEM images showed that the pore size of GelMA decreased with increasing concentration (e.g. Figure 3 Larger pores accommodate more cells than smaller pores, promoting cell contact. Immunofluorescence images showed no difference in cell spreading on the first day. On day 4, the aspect ratio of cells in GelMA hydrogels decreased with increasing GelMA concentration, indicating that 7.5% and 5% GelMA can provide better scaffolds for cell spreading (as shown in Figure 2A and B). Figure 3 C and D in the figure).

[0069] Rheometer tests showed that G' of the three concentrations of GelMA hydrogels was always greater than G' under different frequencies (0-100 rad / s), temperatures (10-40 °C) and times (0-3 min). Mechanical tests performed by a universal material testing machine showed that the maximum compressive strength and Young's modulus increased with increasing GelMA concentration. Microfibers made of 5% GelMA were brittle and easily broken. 7.5% and 10% GelMA were more conducive to the formation of microfibers and maintained a stable morphology. Considering the cell diffusion and mechanical properties of the three concentrations of GelMA, 7.5% GelMA could be selected as the scaffold material for cell-laden microfibers.

[0070] 2. Exploration of cell concentration:

[0071] In an embodiment of the present invention, GelMA was injected into a transparent silicon tube (400 μm in diameter and 10 cm in length) and exposed to purple light for 30 seconds for photocrosslinking. The manufactured microfibers were extruded from the silicon tube. The embodiment of the present invention found that the microfibers were soft microfibers with a diameter of about 400 μm. The microfibers are highly elastic and can be stretched to twice their original length and then restored to their original length without obvious damage in the process. SEM images show that the surface of the microfibers is full of micropores with diameters of 21.71 μm to 65.39 μm. These pores are interconnected within the microfibers, not only forming channels for the exchange of nutrients and waste, but also providing space for cells to extend, migrate and interact.

[0072] In order to explore the appropriate cell concentration, the present invention designed three cell concentrations (0.5×10 7 cell / mL, 1×10 7 cell / mL and 2×10 7 cell / mL), and three different concentrations of HUVEC-loaded microfibers and DPSC-loaded microfibers were prepared respectively. Specifically, the centrifuged cell pellets were resuspended in the GelMA solution according to the corresponding concentration and fully mixed. The cell-loaded GelMA solution was injected into a transparent silicon tube, and the cell-loaded microfibers were prepared in the same manner as described above.

[0073] The results showed that cells were evenly distributed in the microfibers and proliferated over time. Cells could migrate from the interior to the surface of the microfibers, and as the cells spread, the microfibers changed from being initially smooth to being slightly concave. Implanting more cells was beneficial for tissue formation, but too high a cell concentration (2×10 7 The microfibers containing 1×10 7 The microfibers containing 1×10 DPSC and HUVEC were as stable as those containing lower cell concentrations. 7 The subsequent experiments were carried out with 10 cells / mL.

[0074] 3. Evaluation of cell activity and cell characteristics loaded in microfibers:

[0075] In an embodiment of the present invention, the microfibers loaded with DPSC and the microfibers loaded with HUVEC were cultured for 7 days and measured on the 1st, 4th and 7th days of culture to evaluate cell viability. Live and dead cells were identified using a live / dead survival rate kit (KeyGen, China). The working solution was diluted with a PBS solution containing 0.5‰ calcein AM (green) and 0.5‰ propidium iodide (PI, red). The microfibers loaded with cells were washed with PBS and incubated in the working solution at room temperature for 30 minutes to prevent light exposure. After incubation, the samples were washed three times with PBS, anti-fluorescence quenching sealing liquid was added, and pictures were collected with CLSM. The number of live and dead cells was calculated by ImageJ software. Figure 4The results of the viability and proliferation ability of the cells loaded in the microfibers prepared in the examples of the present invention are shown, wherein part A shows the fluorescence images of the live / dead determination of DPSCs in the microfibers on days 1, 4 and 7 (scale bar, 200 μm). Part B shows the fluorescence images of Ki-67 (red) and DAPI (blue) staining of DPSCs in the microfibers (scale bar, 200 μm). Part C shows the quantification of the viability of DPSCs in the microfibers on days 1, 4 and 7. Part D shows the fluorescence images of the live / dead determination of HUVECs in the microfibers on days 1, 5 and 7 (scale bar, 200 μm). Part E shows the fluorescence images of Ki-67 (red) and DAPI (blue) of HUVECs in the microfibers (scale bar, 200 μm). Part F shows the quantification of the viability of HUVEs in the microfibers on days 1, 4 and 7.

[0076] In the present example, a live / dead assay was performed to investigate the activity of DPSCs and HUVECs in GelMA microfibers. The viability of DPSCs and HUVECs was analyzed on days 1, 4, and 7 of culture. As shown in the CLSM images, only a small number of dead cells (e.g., Figure 4 Quantitative analysis showed that DPSCs in GelMA microfibers maintained the lowest viability (89.08% ± 9.08%) on day 1 and the highest viability (95.78% ± 3.47%) on day 4 (as shown in Part A and Part D of Figure 2). Figure 4 On the 7th day of culture, the viability of DPSCs decreased slightly, but still remained at a high level (94.47% ± 3.01%). On the 1st, 4th and 7th days of culture, the survival rate of HUVECs in microfibers gradually increased from 86.53% ± 4.06% to 90.31% ± 2.55% and 96.48% ± 3.07% (as shown in Figure 5C). Figure 4 These results indicate that GelMA microfibers have low cytotoxicity and good biocompatibility, and can provide a good environment for the survival of DPSCs and HUVECs.

[0077] The proliferation capacity of cells in microfibers is very important for cell expansion and tissue formation. Ki67 is an antigen associated with cell proliferation and is used as an indicator of the proliferation capacity of DPSC and HUVEC within 7 days of culture. In the present embodiment, two types of microfibers were stained with Ki-67 (Abcam, UK) at a dilution of 1:200 to detect cell proliferation on days 1, 4, and 7 of culture. The results of Ki-67 staining were consistent with the live / dead cell assay, showing that the number of live cells increased after culture (e.g. Figure 4(shown in part B). Ki-67 immunofluorescence staining of DPSC-loaded microfibers showed that most cells expressed Ki-67 during in vitro culture, indicating the strong vitality of DPSC and HUVEC in the microfibers. During in vitro culture, the present invention observed that HUVECs had good fluidity in GelMA microfibers, and a portion of HUVECs moved out of the microfibers during the culture process. Since the migration of HUVECs is a necessary prerequisite for angiogenesis, this microfiber property that allows HUVEC migration can promote rapid regeneration of blood vessels.

[0078] Functional cells in supporting scaffolds are a prerequisite for tissue formation. In order to explore whether DPSCs and HUVECs in microfibers can work properly, the cells were stained with several characteristic biomarkers. Specifically, DPSC-loaded microfibers and HUVEC-loaded microfibers were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.2% Triton X-100 for 15 minutes. Afterwards, the microfibers were blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich, USA) solution for 30 minutes, and all antibodies were diluted with 1% BSA. On days 1, 4, 7, and 14 of culture, the DPSC-loaded microfibers were fixed and incubated in primary antibodies of laminin (LN; Abcam, UK) and CD90 (Zen BioScience, China) at a dilution of 1:200 overnight at 4 °C, while the HUVEC-loaded microfibers were harvested on days 1, 4, 7, and 14 and stained in the same manner with primary antibodies of VE-cad (Abcam, UK) and CD31 (Novus Biologicals, USA). The samples were incubated for 2 hours in dark conditions using Alexa Fluor 555 (1:500 dilution, Invitrogen, USA) and phalloidin (1:1000 dilution, Invistrogen). The nuclei of the cells were stained with DAPI for 10 minutes. After each incubation step, the samples were gently washed three times with PBS solution. The stained microfibers were imaged using CLSM. The results are shown in Figure 2. Figure 5 As shown, Figure 5 The results of cell staining fluorescence images of the microfibers prepared in the examples of the present invention are shown. Including: fluorescence images of DPSCs in GelMA microfibers stained with DAPI (blue), F-actin (green), CD90 (red) and laminin (red) on days 1, 4, 7 and 14 (scale bar, 200 μm). Fluorescence images of HUVECs in GelMA microfibers stained with DAPI (blue), F-actin (green), CD31 (red) and VE-cad (red) on days 1, 4 and 7.

[0079] CD90 is a stem cell marker that is closely related to the regenerative capacity of stem cells. Immunofluorescence analysis showed that a high proportion of DPSCs positively expressed CD90 in microfibers on days 1, 4, 7, and 14 of culture, indicating that DPSCs in microfibers can maintain cell stemness (e.g., Figure 5 As shown in part A of the figure). Laminin plays an important role in cell adhesion and movement. Immunofluorescence staining showed that most DPSCs in the microfibers could positively express laminin, indicating that DPSCs could play a normal adhesion and migration function in GelMA microfibers (as shown in part A of the figure). Figure 5 F-actin immunostaining showed that the cytoskeleton of DPSCs gradually unfolded and the cell aspect ratio increased over time (as shown in Figure 2A). Figure 4 (as shown in parts A and B in the figure).

[0080] CD31 is a specific marker of HUVEC and is associated with angiogenesis. VE-cad is associated with HUVEC proliferation and angiogenesis. Fluorescence images showed that CD31 and VE-cad were positively expressed in most HUVECs on days 1, 4, and 7 of culture (e.g. Figure 4 The results show that HUVECs maintain a strong angiogenesis ability in GelMA microfibers. The present invention embodiment noted that after 7 days of culture, the number of cells stained with F-actin decreased (as shown in Figure 2A). Figure 4 Close inspection of the plates revealed that most of the HUVECs migrated from the microfibers to the bottom of the plates, explaining the decrease in cells in the microfibers.

[0081] 4. Permeability evaluation of GelMA microfiber aggregates (MA):

[0082] In an embodiment of the present invention, in order to achieve pulp regeneration, microfibers containing DPSC and HUVEC are mixed to produce microfiber aggregates (MA), and MA has a loose three-dimensional network. The permeability of the material promotes the transport of nutrients and oxygen from the host to the transplant, which is essential for cell survival. Therefore, the liquid permeability between MA and hydrogel blocks is compared in an embodiment of the present invention. In an embodiment of the present invention, the liquid diffusion capacity of MA is tested by measuring the permeation rate of eosin dye solution under gravity and comparing it with the GelMA hydrogel block.

[0083] The present invention found that the dye solution penetrated most of the microfibers almost immediately after the addition of MA. After 30 minutes, the dye solution completely penetrated the MA and flowed to the lowest point of the funnel, and there was no residual dye solution above the microfibers. 30 minutes after adding to the hydrogel block, the dye solution only slightly penetrated the hydrogel. After 90 minutes, most of the volume of the hydrogel had been penetrated, but the entire hydrogel block had not been penetrated, and a large amount of dye solution was above the hydrogel block. The results are shown in Figure 2. Figure 6 As shown, Figure 6 The permeability evaluation results of the GelMA microfiber aggregate (MA) prepared in the embodiment of the present invention are shown. Part A shows the top-down penetration images of the eosin solution in the MA and the hydrogel block at 0, 0.5 and 2 hours. Part B shows the image of the eosin solution penetration in the MA and the hydrogel block under the siphon effect for 5 hours. Part C shows the quantitative analysis of the distance that the eosin penetrates into the MA and the hydrogel block under the siphon effect.

[0084] In theory, the narrow space between the microfibers can induce the flow of liquid through capillary action, thereby promoting the entry of body fluids into the interior of the microfibers. In the embodiment of the present invention, the penetration distance of the eosin dye solution under the siphon effect in MA and hydrogel blocks was quantitatively evaluated. MA and hydrogel blocks were located at the tip of a pipette with a small hole at the tip to simulate the tooth root. After the tip of the pipette containing the two groups of materials was immersed in the eosin staining solution for 5 hours, the staining solution penetration distance of each sample was observed and calculated. Figure 6 As shown in Part B, the liquid penetration distance of MA is longer than that of the hydrogel block. According to calculations, the penetration distance of the control group is 4.83±0.62mm, while the penetration distance of MA is 8.33±0.62mm (as shown in Figure 2). Figure 6 The results of both experiments showed that the fluid permeability of MA was greater than that of the hydrogel block, which helped the transplanted cells to obtain nutrients and oxygen.

[0085] Example 2: Subcutaneous ectopic dental pulp regeneration experiment in nude mice

[0086] In the embodiment of the present invention, human tooth roots are collected from extracted human premolars, the middle part of the tooth root is selected with a dental handpiece, and it is ground into a root segment (RS) of 5-6 mm, the cementum pulp is removed, and the root segment is ground into an approximately elliptical cross-section with a long diameter of 3 mm and a short diameter of 2 mm to obtain a root cannula, which is similar to the shape of a natural root. The root cannula is then placed in sterile PBS and ultrasonically cleaned for 10 minutes in an ice bath, and repeated three times. Then, 17%, 10% and 5% ethylenediaminetetraacetic acid (EDTA, KESHI) are ultrasonically cleaned for 10 minutes respectively. The larger end of the root cannula is sealed with Iroot BP (Iroot, Canada) to imitate a natural tooth root.

[0087] In the examples of the present invention, the in vivo experiments were divided into two groups: cell-loaded microfibers (MA group) and cell-loaded hydrogel blocks. Immediately after the preparation of the microfibers, microfibers loaded with DPSCs and microfibers loaded with HUVECs (length 5 cm) were added to the root cannula in a ratio of 2:1. DPSCs and HUVECs were mixed in a GelMA solution at a ratio of 2:1, injected into the root cannula, and cross-linked to prepare a cell-loaded hydrogel block as a control group. For the control group, the volume of the GelMA solution was the same as that of the microfiber group. After 4 weeks of in vitro culture, the root cannulas with these cell-loaded scaffolds were subcutaneously implanted into the back of immunodeficient nude mice. Each mouse carried 4 root cannulas. After 8 weeks of implantation, the mice were killed and the root cannulas were obtained for analysis.

[0088] In an embodiment of the present invention, the harvested root cannula samples were fixed in 4% formaldehyde overnight, decalcified in 17% EDTA for 2 months, embedded in paraffin blocks, and then cut into 5 μm thick sections. The two groups of sections were stained with hematoxylin and eosin (HE) staining solution to evaluate the morphology and structure of the regenerated pulp-like tissue. In order to observe collagen formation in the newly formed tissue, the samples were stained with Masson (Basso, China) according to the specifications. Immunohistological staining was performed using antibodies against dentin matrix protein 1 (DMP-1, Santa Cruz, USA), dentin salivary phosphoprotein (DSPP; Santa Cruz, US) and Nestin (DSPP, Santa Cruz, US). The cells stained brown near the dentin wall were counted and determined to be DMP-1 positive cells. Tissue with an area of ​​approximately 0.025 mm2 near the dentin was selected for statistics. The differences between the two groups were analyzed by T test. The results are as follows. Figure 7 As shown, Figure 7 The results of the evaluation of the new tissue of ectopic pulp regeneration in the embodiment of the present invention are shown, wherein Part A shows a schematic diagram of the experimental process of ectopic pulp regeneration of cell-containing GelMA microfiber aggregates and cell-containing GelMA hydrogel blocks in the embodiment of the present invention. Part B is a HE staining result of pulp-like tissue regeneration in the two groups of samples (scale bar, 500 μm). Part C is an enlarged image of the coronal, middle and top pulp-like tissues of the two groups of samples (scale bar, 40 μm). Part D is a quantitative analysis of the ratio of the pulp tissue area to the inner wall area of ​​the root cannula. Part E is the HE and Masson staining results of the dentin-pulp composite structure of the two groups, wherein the MA group detected a new dentin-like structure (green square), but the control group did not detect it (scale bar, 50 μm). Part F shows the quantitative statistics of the number of cells positively expressing DMP-1 (scale bar, 50 μm), wherein the immunohistochemical staining of DMP-1 and DSPP showed positive expression in some cells. Part G shows the statistics of the number of DMP-1 and DSPP positive cells.

[0089] In the present embodiment, the HE images of the in vivo samples showed that the regenerated pulp-like tissue of the control group was mainly located at the top 2 / 3 of the tooth root, while the regenerated pulp-like tissue of the MA group almost filled the entire root cannula (e.g. Figure 7 Quantitative calculations showed that the area of ​​regenerated tissue covered 37% ± 2.42% of the root cannula in the control group and 72% ± 3.92% in the MA group (as shown in Figure 2A). Figure 7 In the embodiment of the present invention, the two groups of samples were equally divided into coronal, middle and apical slices, and the same position was selected from each slice for amplification (as shown in part D). Figure 7 In the middle and apical sections of the control group, dense pulp tissue was observed, while the coronal section was empty, while the three sections of the MA group were filled with dense pulp tissue. HE staining showed that the root canal wall of the MA group formed a structure similar to new dentin (green square). Immunohistochemical staining showed that odontoblast markers DMP-1 and DSPP were expressed in small amounts in cells close to the dentin wall (as shown in Figure 2A). Figure 7 The number of positive cells in the two groups was counted and the analysis showed that there was a statistical difference in the expression of DMP-1 and DSPP between the two groups (as shown in Figure 5A). Figure 7 (as shown in part G in the figure).

[0090] These results suggest that MA was able to regenerate more tissue compared with hydrogel blocks and was effective in inducing DPSCs to differentiate into odontoblast-like cells. The MA group regenerated dentin-like tissue, which is expected to promote further development of tooth roots.

[0091] Figure 8 The results of angiogenesis in the ectopic dental pulp regeneration test in the embodiment of the present invention are shown. Part A shows the image and quantitative analysis of the blood flow at the opening of the dentin fragment by laser Doppler blood flowmetry. Part B shows the image of the microvessels at the top opening of the root (green arrow). Part C shows the HE staining results of two groups of regenerated blood vessels (green arrow) (scale bar, 100μm). Part D shows the fluorescent images (green arrows) of CD31 (red) and DAPI (blue) of the extraluminal cells (scale bar, 100μm). Part E shows the per unit area (1mm 2 ) Quantitative analysis of the number of regenerated blood vessels.

[0092] Neovascularization is a prerequisite for successful tissue regeneration. The present invention compares angiogenesis between MAs and a control group by laser Doppler blood flow measurement and histological analysis. The perfusion unit (PU) reading detected by laser Doppler blood flow measurement can directly reflect the local microcirculatory blood flow, which is a reliable signal for revascularization. Laser Doppler blood flow map analysis showed that the average value of the control group was 240.22±10.43PU, and the MA group was 310.128±18.55PU. The PU value of the MA group was significantly higher than that of the control group (such as Figure 8 Before collecting samples, we observed obvious blood vessels at the apical opening in the MA group but not in the control group (as shown in Figure 2A). Figure 8 HE staining showed extensive angiogenesis in the regenerated tissue of the MA group (as shown in part B). Figure 8 To further verify the presence of blood vessels, immunofluorescence staining was performed. Fluorescence images showed that cells surrounding the lumen positively expressed CD31, a specific marker for endothelial cells, further supporting the formation of blood vessels (as shown in Figure 2A). Figure 8 The number of blood vessels per square millimeter was calculated in the microfiber group and the control group. The results showed that the number of blood vessels in the MA group was 12.93±2.83, while that in the control group was 4.96±0.48 (as shown in Figure 2). Figure 8 (shown in part E).

[0093] Regenerative tissue cell source test:

[0094] After transplantation, cells from the host's surroundings may also migrate into the dental canal through the root apex. In order to determine whether the implanted DPSCs and HUVECs regenerated dental pulp tissue, the present invention used human mitochondrial antibodies for immunofluorescence identification, which only labeled the mitochondria of human cells, but not the mitochondria of mouse cells. Mouse skeletal muscle was used as a negative control, which confirmed the specificity of the antibody for human cells. The results are shown in Figure 2. Fig. 9 As shown, Fig. 9 The results of evaluating the cell sources of regenerated dental pulp tissue and blood vessels in the embodiment of the present invention are shown. Part A shows a fluorescent image of human mitochondria (green) and CD90 (red) co-stained in the dental pulp tissue during transplantation (scale bar, 20 μm). Part B shows an immunofluorescence image of human mitochondria (green) and CD31 (red) co-stained in the blood vessels during transplantation (scale bar, 50 μm).

[0095] The results showed that some cells in the control group and MA group co-expressed CD90 and human mitochondrial antigen, while cells in mouse muscle did not express human mitochondrial antigen. This means that the regenerated tissue cells originated from the implanted human DPSCs and the host (such as Fig. 9In addition, cells surrounding the blood vessels co-expressed human mitochondria and CD31, indicating that the blood vessels were formed by implanted HUVECs (as shown in Figure 2A). Fig. 9 (shown in part B).

[0096] In the embodiment of the present invention, the cell-loaded GelMA microfibers prepared by the silicon tube-based coagulant-free bath method have excellent cell compatibility and provide a good environment for cell growth. DPSCs and HUVECs loaded in GelMA microfibers have high cell viability and strong proliferation ability, and can actively express their characteristic biomarkers. In the subcutaneous ectopic dental pulp regeneration experiment of nude mice, TRs loaded with DPSCs-loaded microfibers and HUVECs-loaded microfibers were able to regenerate extensively vascularized pulp tissue and newly formed dentin structure. A small number of cells positively expressed DMP-1 and DSPP, indicating the formation of odontoblast-like cells. The embodiment of the present invention confirms that cell-loaded GelMA microfiber aggregates are a new solution for vascularized dental pulp regeneration, which can be used as root canal implants to promote the clinical transformation and application of dental pulp regeneration.

[0097] For the method embodiments, for the sake of simplicity, they are all described as a series of action combinations, but those skilled in the art should know that the present invention is not limited by the order of the actions described, because according to the present invention, some steps can be performed in other orders or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and components involved are not necessarily required by the present invention.

[0098] The above is a detailed introduction to a cell-laden microfiber, a preparation method, a root canal implant and an application provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for a person skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A cell-laden microfiber aggregate, characterized in that: The cell-loaded microfiber aggregate comprises: cell-loaded microfibers, which are methacrylated gelatin microfibers loaded with dental pulp stem cells and methacrylated gelatin microfibers loaded with human umbilical vein endothelial cells; the cell-loaded microfibers are used to imitate the structure of dental pulp; The cell content in the cell-laden microfibers is 0.5*107cell / ml~2*107cell / ml; The cell-laden microfiber has a diameter of 300-500 μm and a length of 5 cm; The preparation method of the microfiber aggregate comprises: Prepare dental pulp stem cells or human umbilical vein endothelial cells; The dental pulp stem cells or human umbilical vein endothelial cells are respectively mixed with the methacrylated gelatin solution to obtain a matrix solution; The matrix solution is injected into a transparent silicone tube, and then the transparent silicone tube is exposed to ultraviolet light for 30 seconds; the matrix solution gradually cross-links in the transparent silicone tube to form microfibers, and then the microfibers are pushed out with a needle of appropriate diameter to obtain the cell-carrying microfibers. After obtaining microfibers carrying dental pulp stem cells and microfibers carrying human umbilical vein endothelial cells respectively, the two are mixed and disorderly entangled to obtain microfiber aggregates.

2. The cell-laden microfiber aggregate according to claim 1, characterized in that: The concentration of the methacrylated gelatin solution in the cell-laden microfiber aggregate is 50-100 mg / ml.

3. A method for preparing the cell-laden microfiber aggregates according to claim 1, characterized in that: The method comprises: Prepare dental pulp stem cells or human umbilical vein endothelial cells; The dental pulp stem cells or human umbilical vein endothelial cells are respectively mixed with the methacrylated gelatin solution to obtain a matrix solution; The matrix solution is injected into a transparent silicone tube, and then the transparent silicone tube is exposed to ultraviolet light for 30 seconds; the matrix solution is gradually cross-linked in the transparent silicone tube to form microfibers; The microfiber is pushed out of the transparent silicone tube to obtain the cell-carrying microfiber, and then the microfiber is pushed out with a needle of suitable diameter to obtain the cell-carrying microfiber, and after obtaining microfibers carrying dental pulp stem cells and microfibers carrying human umbilical vein endothelial cells respectively, the two are mixed and the two are disorderly entangled to obtain microfiber aggregates; The cell content in the cell-carrying microfiber is 0.5*107cell / ml~2*107cell / ml; the diameter of the cell-carrying microfiber is: 300-500μm.

4. A root canal implant, characterized in that: It comprises a cell-loaded microfiber aggregate composed of two types of cell-loaded microfibers, and the cell-loaded microfiber aggregate is the cell-loaded microfiber aggregate according to claim 1 or 2.

5. The root canal implant according to claim 4, characterized in that: In the root canal implant, the ratio of methacrylated gelatin microfibers loaded with dental pulp stem cells to methacrylated gelatin microfibers loaded with human umbilical vein endothelial cells is 2:1.

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