Stem cell-free multilayered microtissue spheroids for dental pulp regeneration and methods of making and using the same

Abstract

The application provides a scaffold-free multicellular microtissue sphere for dental pulp regeneration and a preparation method and application thereof, and belongs to the technical field of biomaterials. The scaffold-free multicellular microtissue sphere is a dense sphere formed by self-assembly of dental pulp stem cells, endothelial-like cells and / or neural-like cells through cell-cell interaction. The endothelial-like cells and neural-like cells are obtained by inducing differentiation of the dental pulp stem cells. The dental pulp stem cells are co-cultured with the autologous endothelial-like cells and / or neural-like cells, and a tight connection is formed by cell-cell interaction (such as collagen secretion), so that a three-dimensional microtissue sphere is self-assembled. The microtissue sphere better simulates the in-vivo microenvironment, can promote the vascularization and innervation of regenerated dental pulp under the condition of no external scaffold, and can avoid the immune problems caused by the external scaffold and non-autologous cells.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a scaffold-free multicellular microsphere for dental pulp regeneration, its preparation method, and its application. Background Technology

[0002] Dental pulp is loose connective tissue located within the root canal system of a tooth, responsible for nutrient supply, sensory conduction, and dentin formation. Irreversible damage to the dental pulp can occur due to caries, trauma, and other causes. While the current standard clinical treatment—root canal therapy—can remove infection and preserve the tooth, it leads to permanent loss of pulp function, making the tooth brittle and prone to fracture. Pulp engineering aims to replace damaged pulp with regenerated pulp-like tissue, restoring its physiological function and representing the ultimate goal of regenerative oral medicine.

[0003] Ideal pulp regeneration should achieve the integrated reconstruction of the pulp-dentin complex, vascular network, and neural network. Timely vascular reconstruction is the primary prerequisite for the survival and function of implanted cells, while the restoration of neural innervation is crucial for the sensory and defensive functions of the pulp. Therefore, how to synergistically promote the vascularization and neuralization of regenerated pulp is a core challenge in this field.

[0004] In existing technologies, one strategy for constructing vascularized dental pulp tissue is to co-culture dental pulp stem cells and endothelial cells. However, allogeneic endothelial cells pose a risk of immune rejection, are difficult to obtain from their own source, and have limited in vitro expansion capabilities, thus hindering clinical translation. Another strategy involves using scaffold materials such as hydrogels to load cells and growth factors, but issues such as degradation products of exogenous scaffolds, implantation difficulties, and potential inflammatory reactions remain to be addressed. Furthermore, existing technologies have proposed strategies for loading stem cells onto injectable microcarriers and inducing their differentiation through sustained-release drugs. However, this method still relies on exogenous scaffolds, suffers from uncertainties in degradation products and potential inflammatory risks, and cannot achieve in vitro pre-assembly into active microtissues with both vascularization and neuralization functions, thus limiting regeneration efficiency and integrity.

[0005] Therefore, there is a need for a material for pulp regeneration that does not require a scaffold, avoids immune rejection, and does not require degradation. Summary of the Invention

[0006] In view of this, the present invention provides a scaffold-free multicellular microtissue sphere for dental pulp regeneration that does not require a scaffold, avoids immune rejection, and does not require degradation.

[0007] It is also necessary to provide a method for preparing scaffold-free multicellular microtissue spheres for dental pulp regeneration.

[0008] There is also a need to provide an application of scaffold-free multicellular microtissue spheres for pulp regeneration.

[0009] The technical solution adopted by this invention to solve its technical problem is:

[0010] A scaffold-free multicellular microtissue sphere for dental pulp regeneration, wherein the scaffold-free multicellular microtissue sphere is a dense sphere formed spontaneously by dental pulp stem cells, endothelial-like cells and / or neuron-like cells through intercellular interactions, wherein the endothelial-like cells and neuron-like cells are obtained by induced differentiation of dental pulp stem cells.

[0011] Preferably, the ratio of the number of dental pulp stem cells, endothelial-like cells, and neuron-like cells is 2-10:0-5:0-5, and the number of endothelial-like cells and neuron-like cells is not simultaneously 0.

[0012] Preferably, the ratio of dental pulp stem cells, endothelial-like cells, and neuron-like cells is one of 1:1:1, 3:1:1, 1:1:0, 3:1:0, 1:0:1, and 3:0:1.

[0013] A method for preparing scaffold-free multicellular microtissue spheres for dental pulp regeneration as described above includes the following steps:

[0014] S1 Obtaining endothelial-like cells and / or neuron-like cells derived from DPSCs: Dental pulp stem cells were cultured in endothelial cell induction medium and / or neuron cell induction medium, respectively, and induced to differentiate into endothelial-like cells and / or neuron-like cells, respectively, to obtain endothelial-like cells and / or neuron-like cells.

[0015] S2 constructs a multi-cell co-culture system: DPSCs, endothelial-like cells and / or neuron-like cells are mixed and seeded in a low-adhesion culture plate;

[0016] Culture and formation of S3 micro-tissue spheres: The cells were continuously cultured in an incubator at 37℃ and 5% CO2. During this period, the cells spontaneously aggregated and formed spherical, scaffold-free, three-dimensional multicellular micro-tissue spheres.

[0017] Preferably, the endothelial-like cells and neuron-like cells obtained from DPSCs in step S1 need to be cultured for 10-14 days.

[0018] Preferably, in step S1, endothelial-like cells and nerve-like cells derived from DPSCs are obtained, and the endothelial cell induction culture medium is a basal culture medium supplemented with basic fibroblast growth factor, dexamethasone, vascular endothelial growth factor and bovine serum albumin.

[0019] Preferably, the nerve cell induction culture medium is a basic nerve culture medium supplemented with basic fibroblast growth factor, epidermal growth factor and B27 additive.

[0020] Preferably, in step S3, the culture is carried out in an incubator for more than 3 days.

[0021] The application of scaffold-free multicellular microspheres for pulp regeneration as described above in pulp tissue regeneration grafts promotes vascularization and / or neuralization of pulp tissue regeneration.

[0022] Preferably, the graft is used for implantation into the pulp chamber or root canal after pulpectomy.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention provides a scaffold-free multicellular micro-tissue sphere for dental pulp regeneration. The scaffold-free multicellular micro-tissue sphere is a dense sphere formed spontaneously by dental pulp stem cells, endothelial-like cells, and / or neuron-like cells through intercellular interactions. The endothelial-like cells and neuron-like cells are obtained by induced differentiation of dental pulp stem cells. This invention co-cultures dental pulp stem cells with autologous endothelial-like cells and / or neuron-like cells, relying on intercellular interactions (such as collagen secretion) to form tight junctions and spontaneously aggregate to form three-dimensional micro-tissue spheres. This better simulates the in vivo microenvironment and can simultaneously promote the vascularization and neuralization of regenerated dental pulp without exogenous scaffolds, while avoiding the immune problems caused by exogenous scaffolds and non-autologous cells. Attached Figure Description

[0025] Figure 1 The images show primary and passaged DPSCs under an inverted microscope. (A) Primary cells crawl out of the adherent tissue block after three days. (B) First-generation DPSCs after three days of passage.

[0026] Figure 2 This is a microscopic image of endothelial-like cells of DPSCs after 14 days of endothelial induction.

[0027] Figure 3 Microscopic observation of DPSCs and Matrigel tube formation of endothelial-like cells, (A) DPSCs group; (B) endothelial-like cell group.

[0028] Figure 4 Bar chart for quantitative indicators of angiogenesis in DPSCs and endothelial-like cells (percentage of vessel area) (**** indicates P<0.0001).

[0029] Figure 5 Bar chart showing the relative mRNA expression levels of VEGF, a marker gene for angiogenesis in DPSCs and endothelial-like cells (*** indicates P<0.001).

[0030] Figure 6 Giemsa staining of neuron-like cells obtained after 14 days of DPSC neural induction; arrows indicate neural processes.

[0031] Figure 7 Bar chart showing the relative mRNA expression levels of the neurogenic marker gene NCAM in DPSCs and neuron-like cells (* indicates P<0.05).

[0032] Figure 8 These are gross observation images of DPSC-derived cell microspheres from Examples 1-6 and Comparative Example 1. (A) Growth status of DPSC-derived cell microspheres after 3 days of culture in a low-adhesion 96-well plate. Cells aggregated into clusters, and all seven groups of cell microspheres were round or oval. Arrows indicate the seven groups of cell microspheres respectively; (B) Transferring DPSC-derived cell microspheres to a blue sterile cloth using a pipette, their morphology remained largely unchanged. The figure shows the seventh group of DPSC-derived cell microspheres (all constructed from DPSCs).

[0033] Figure 9 HE staining images of DPSC-derived cell microspheres: (AG) HE staining images of DPSC-derived cell microspheres from group 1 to group 7; (HN) HE staining images of the central portion of DPSC-derived cell microspheres from group 1 to group 7.

[0034] Figure 10 Masson staining images of DPSC-derived cell microspheres. Arrows indicate collagen fibers. (AG) Masson staining images of DPSC-derived cell microspheres in groups 1 through 7. (HN) Masson staining images of the central portion of DPSC-derived cell microspheres in groups 1 through 7.

[0035] Figure 11 The images show the CD31 immunofluorescence staining patterns of seven groups of DPSC-derived cell microspheres. (AG) represent the CD31 immunofluorescence staining patterns of the first to seventh groups of DPSC-derived cell microspheres (blue: DAPI, red: CD31).

[0036] Figure 12 The bar chart shows the average fluorescence intensity of CD31 in cell microspheres derived from DPSCs for Examples 1-6 and Comparative Example 1 (**** indicates P<0.0001, *** indicates P<0.001, * indicates P<0.05).

[0037] Figure 13 The images show Nestin immunohistochemical staining images of DPSC-derived cell microspheres from Examples 1-6 and Comparative Example 1. (AG) are Nestin immunohistochemical staining images of DPSC-derived cell microspheres from Groups 1 to 7, respectively (blue: DAPI, brown: Nestin).

[0038] Figure 14 Bar charts showing the percentage of Nestin-positive cell area in cellular microspheres derived from DPSCs for Examples 1-6 and Comparative Example 1 (**** indicates P<0.0001).

[0039] Figure 15 Examples 1-6 and Comparative Example 1 show confocal microscopic views of live and dead cell staining of DPSC-derived cell microspheres. (AG) are confocal microscopic images of live and dead cell staining in groups 1 to 7, respectively (green: live cells, red: dead cells).

[0040] Figure 16 Bar charts showing the number of dead cells in cell microspheres derived from DPSCs in Examples 1-6 and Comparative Example 1 (**** indicates P<0.0001).

[0041] Figure 17 Examples 1-6 and Comparative Example 1 illustrate the surgical procedure for subcutaneous heterotopic transplantation of DPSC-derived cell microtissue spheres in nude mice. (A) The cell microtissue spheres were aspirated from the culture plate using a pipette; (B) The cell microtissue spheres were placed on a sterile weighing spoon; (C) A subcutaneous bag was placed on the back of the nude mouse; (D) A rabbit molar filled with cell microtissue spheres was implanted into the subcutaneous bag; (E) The wound was closed and sutured.

[0042] Figure 18 Gross observations after ectopic transplantation of DPSC-derived cell microspheres and blank control group in Examples 1-6, Comparative Example 1, and the blank control group. (AC) Rabbit molars under the skin of nude mice, showing rabbit molars filled with the first group of DPSC-derived cell microspheres; (D) Gross view of rabbit molars filled with the first to fifth groups of cell microspheres 8 weeks after ectopic transplantation; (E) Gross view of rabbit molars filled with the sixth and seventh groups of cell microspheres 8 weeks after ectopic transplantation; (F) Gross view of rabbit molars filled with calcium hydroxide paste (blank control group) 8 weeks after ectopic transplantation.

[0043] Figure 19 HE staining images of seven groups of DPSC-derived cell microspheres and a blank control group after ectopic transplantation. Arrows indicate vascular-like structures. (AH) HE staining images of molars after ectopic transplantation in groups 1 to 7 and the blank control group. (IP) HE staining images of newly formed tissue in the local area after ectopic transplantation in groups 1 to 7 and the blank control group.

[0044] Figure 20 Bar chart showing the number of microvessels after ectopic transplantation of cell tissue spheres in seven groups. (* indicates P<0.05).

[0045] Figure 21Masson staining images of seven groups of DPSC-derived cell micro-tissue spheres and a blank control group after ectopic transplantation. Red arrows indicate vascular-like structures, blue arrows indicate collagen fiber-like structures, yellow arrows indicate odontoblasts (I, M, N, O are magnified views of the cells in the upper right corner), and green arrows indicate necrotic foci. (AH) Masson staining images of molars after ectopic transplantation in groups 1 to 7 and the blank control group. (IP) Masson staining images of newly formed tissue in groups 1 to 7 and the blank control group after ectopic transplantation.

[0046] Figure 22 Immunohistochemical staining images of CD31 in molars after ectopic transplantation of seven groups of DPSC-derived cell micro-tissue spheres and a blank control group. (AH) Immunohistochemical staining images of molars after ectopic transplantation of groups 1 to 7 and the blank control group. (IP) Immunohistochemical staining images of CD31 in newly formed tissue in groups 1 to 7 and the blank control group after ectopic transplantation. (Blue: DAPI, Brown: CD31)

[0047] Figure 23 Bar chart showing the percentage area of ​​CD31-positive cells in seven groups of cell microspheres derived from DPSCs.

[0048] Figure 24 The images show Nestin immunohistochemical staining images of molars after ectopic transplantation of seven groups of DPSC-derived cell micro-tissue spheres and a blank control group. (AH) Nestin immunohistochemical staining images of molars after ectopic transplantation of groups 1 to 7 and the blank control group. (IP) Nestin immunohistochemical staining images of newly formed local tissue after ectopic transplantation of groups 1 to 7 and the blank control group. (Blue: DAPI, Brown: Nestin)

[0049] Figure 25 Bar chart showing the percentage of Nestin-positive cell area in seven groups of cellular microspheres derived from DPSCs.

[0050] Figure 26 Immunohistochemical staining images of GFAP in molars after ectopic transplantation of seven groups of DPSC-derived cell micro-tissue spheres and a blank control group. (AH) Immunohistochemical staining images of molars after ectopic transplantation of groups 1 to 7 and the blank control group. (IP) Immunohistochemical staining images of newly formed tissue with GFAP in groups 1 to 7 and the blank control group after ectopic transplantation. (Blue: DAPI, Brown: GFAP)

[0051] Figure 27 Bar chart showing the percentage of GFAP-positive cell area in seven groups of cellular microspheres derived from DPSCs.

[0052] In the figure: Groups 1 to 7 are Examples 1-6 and Comparative Example 1, respectively. Detailed Implementation

[0053] To facilitate understanding of this application, a more comprehensive description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are also given. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to enable a more thorough and complete understanding of the disclosure of this application.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0055] A scaffold-free multicellular microtissue sphere for dental pulp regeneration, wherein the scaffold-free multicellular microtissue sphere is a dense sphere formed spontaneously by dental pulp stem cells (DPSCs), endothelial-like cells and / or neuron-like cells through intercellular interactions, wherein the endothelial-like cells and neuron-like cells are obtained by induced differentiation of dental pulp stem cells.

[0056] DPSCs primarily serve as structural support, functional reserves, and microenvironment regulation, forming the foundation for successful pulp regeneration. Specifically: 1. DPSCs secrete extracellular matrix, enabling scaffold-free assembly. This means they do not rely on any exogenous scaffold materials; the formation of micro-tissue spheres depends entirely on intercellular interactions and the matrix secreted by the cells themselves. Endothelial-like cells primarily form vascular networks, while neuronal-like cells primarily transmit electrical signals; neither are specialized matrix-secreting cells. If endothelial-like cells and neuronal-like cells are co-cultured alone, they cannot form dense, manipulable spheres due to insufficient ECM support such as collagen. Therefore, DPSCs ensure the "mechanical integrity" of the micro-tissue spheres, a prerequisite for transplantation. 2. After implantation, DPSCs differentiate into odontoblast-like cells, directly participating in dentin regeneration. Endothelial-like cells have already differentiated into the endothelial lineage, primarily participating in vascular network reconstruction in vivo; neuronal-like cells have already differentiated into the neural lineage, primarily participating in nerve fiber regeneration. Neither type II nor type III possesses the ability to transdifferentiate into odontoblasts in vivo. Only DPSCs retain multi-directional differentiation potential (especially odontoblast / osteogenous differentiation ability), making them an essential cell source for dentin regeneration. 3. DPSCs exhibit strong paracrine activity, secreting various survival-promoting, proliferation-promoting, and immunomodulatory factors. A cell-cell dialogue exists between DPSCs and predifferentiated cells. Factors such as VEGF, bFGF, and HGF secreted by DPSCs provide essential nutrients and support signals to endothelial-like cells and neuronal-like cells, significantly improving material exchange in the core region of the three-dimensional structure and increasing overall cell survival.

[0057] The invention chooses to obtain endothelial-like cells and neuron-like cells through DPSC-induced differentiation, rather than directly adding heterologous or mature corresponding cells, based on the following key considerations: 1. All constituent cells originate from the same DPSC sample, have completely identical genetic backgrounds, and pose no risk of immune rejection to the host. 2. DPSCs have superior in vitro proliferation capabilities, ensuring the targeted preparation of sufficient quantities of endothelial-like cells and neuron-like cells as needed. In contrast, mature endothelial cells have limited in vitro passage capacity, and mature neurons cannot proliferate at all, failing to meet the cell quantity requirements for clinical applications. 3. Endothelial-like cells and neuron-like cells induced from the same parental DPSCs have surface adhesion molecules that are highly compatible with undifferentiated DPSCs. The three can spontaneously form dense, morphologically complete three-dimensional spheres through "homogeneous" cell recognition and adhesion. Directly adding heterologous mature cells leads to poor cell sorting and adhesion, failing to form stable co-aggregated micro-tissue spheres.

[0058] Furthermore, the ratio of the number of dental pulp stem cells, endothelial-like cells, and neuron-like cells is 2-10:0-5:0-5, and the number of endothelial-like cells and neuron-like cells is not simultaneously 0.

[0059] DPSCs are the main secretory cells of extracellular matrix (ECM) in micro-tissue spheres. Micro-tissue spheres contain abundant collagen fibers, synthesized and secreted by DPSCs, forming the structural framework of the scaffoldless micro-tissue spheres. When the proportion of DPSCs is below the lower limit, the total amount of ECM is insufficient to form a continuous support network between cells, resulting in microspheres failing to form or exhibiting a loose structure, unable to withstand pipetting operations. Endothelial-like cells and neuron-like cells play specific functional roles in promoting angiogenesis and neuronification, respectively, but their ECM secretion capacity is significantly lower than that of undifferentiated DPSCs. When the proportion of either exceeds 50%, two adverse consequences occur: first, the relative proportion of DPSCs decreases, resulting in insufficient total ECM and reduced microsphere mechanical strength; second, excessive favoring of one lineage disrupts the functional balance among the three cell types. When endothelial-like cells exceed 50%, the Nestin-positive area drops sharply; when neuron-like cells exceed 50%, the CD31-positive area drops sharply.

[0060] Furthermore, the ratio of dental pulp stem cells, endothelial-like cells, and neuron-like cells is one of 1:1:1, 3:1:1, 1:1:0, 3:1:0, 1:0:1, or 3:0:1. This utilizes the multi-directional differentiation potential of DPSCs, pre-differentiating DPSCs from the same source into endothelial-like cells and neuron-like cells respectively. These are then mixed with undifferentiated DPSCs in a specific ratio to construct a scaffold-free, multicellular micro-tissue sphere with both "pre-vascularization" and "pre-neuralization" characteristics. This achieves a unity of immunocompatibility and biological function, and the specific cell ratio ensures the stability of the micro-tissue sphere structure and the integrity of its dual functions. Exceeding the above ratio will result in loose microspheres that cannot form a transplantable dense body.

[0061] A method for preparing scaffold-free multicellular microtissue spheres for dental pulp regeneration as described above includes the following steps:

[0062] S1 Obtaining endothelial-like cells and / or neuron-like cells derived from DPSCs: Dental pulp stem cells were cultured in endothelial cell induction medium and / or neuron cell induction medium, respectively, and induced to differentiate into endothelial-like cells and / or neuron-like cells, respectively, to obtain endothelial-like cells and / or neuron-like cells.

[0063] The dental pulp stem cells are induced 3rd-5th generation cells to obtain cells with high purity, good stemness, and vigorous proliferation.

[0064] S2 constructs a multi-cell co-culture system: DPSCs, endothelial-like cells and / or neuron-like cells are mixed and seeded in a low-adhesion culture plate;

[0065] At the time of inoculation, mild enzymes (TrypLE / Accutase) were used for digestion for 2-3 minutes and monitored under a microscope. Since differentiated cells are fragile and traditional trypsin enzymes damage surface adhesion molecules and protrusion structures, mild enzymes were chosen.

[0066] Culture and formation of S3 micro-tissue spheres: The cells were continuously cultured in an incubator at 37℃ and 5% CO2. During this period, the cells spontaneously aggregated and formed spherical, scaffold-free, three-dimensional multicellular micro-tissue spheres.

[0067] Furthermore, the endothelial-like cells and neuron-like cells obtained from DPSCs in S1 need to be cultured for 10-14 days.

[0068] Furthermore, in S1, endothelial-like cells and nerve-like cells derived from DPSCs are obtained, wherein the endothelial cell induction culture medium is a basal culture medium supplemented with basic fibroblast growth factor, dexamethasone, vascular endothelial growth factor and bovine serum albumin.

[0069] Furthermore, the nerve cell induction culture medium is a basic nerve culture medium supplemented with basic fibroblast growth factor, epidermal growth factor and B27 additive.

[0070] Furthermore, the low-adhesion culture plate is an ultra-low-adhesion 96-well plate, with a total cell count of 1×10⁶ cells per well. 2 Up to 1×10 8 Each sphere is formed with a diameter of 400-600 μm, achieving an optimal balance between operability and nutrient penetration.

[0071] Furthermore, in S3, the culture is carried out in an incubator for more than 3 days.

[0072] The application of scaffold-free multicellular microspheres for pulp regeneration as described above in pulp tissue regeneration grafts promotes vascularization and / or neuralization of pulp tissue regeneration.

[0073] Furthermore, the graft is used to implant into the pulp chamber or root canal after pulpectomy.

[0074] Specifically, scaffold-free multicellular micro-tissue spheres are aspirated and directly implanted into the cleaned pulp cavity or root canal for in situ or ectopic transplantation, thereby achieving functional regeneration of the pulp-dentin complex, especially the reconstruction of vascular and neural networks.

[0075] In existing research, the primary obstacle to pulp regeneration is considered to be ischemia / hypoxia. Timely revascularization is a prerequisite for the survival and function of implanted cells. Therefore, research in this field has mainly focused on vascularization strategies—optimizing pro-angiogenic factors, constructing vascularization scaffolds, and co-culturing endothelial cells. In contrast, neuralization is considered a "delayable" or even "spontaneously occurring" secondary event. There are few intervention strategies specifically targeting the restoration of nerve innervation in pulp regeneration research, and there are no precedents for elevating neuralization to the same level as vascularization and designing them in parallel.

[0076] This bias leads to a technological blind spot: when considering improvements to pulp regeneration strategies, those skilled in the art typically think about "how to more effectively promote vascularization." In existing technologies, strategies for actively introducing pre-differentiated neuron-like cells into the pulp regeneration microenvironment to achieve "pre-neuralization" are rarely reported. This invention breaks away from the one-dimensional mindset of "vascularization first," elevating neuralization to an active intervention target of equal importance to vascularization for the first time. Through a pre-vascularization + pre-neuralization strategy, it constructs multicellular micro-tissue spheres with dual characteristics to synergistically promote vascularization and neuralization of the regenerated pulp.

[0077] Secondly, in existing technologies, the optimal microenvironments for in vitro culture of endothelial cells and nerve cells differ significantly. Endothelial cell culture typically requires serum and pro-angiogenic factors such as vascular endothelial growth factor (VEGF). Nerve cell culture typically requires serum-free conditions, B27 supplementation, and neurotrophic factors (such as NGF and BDNF). However, certain components in serum can inhibit neural differentiation or promote glial cell proliferation. Simply mixing endothelial-like cells and nerve-like cells in the same culture medium inevitably leads to a trade-off: under conditions suitable for endothelial cell survival, nerve cells may dedifferentiate or undergo apoptosis; under conditions suitable for nerve cell survival, endothelial cells may lose function. This invention achieves a functional "compromise microenvironment" through precise control of the culture medium ratio—maintaining both CD31 expression and tubule formation ability of endothelial-like cells and Nestin expression and process extension ability of nerve-like cells.

[0078] Finally, existing research reports that different types of cells may exhibit a "sorting effect" when co-cultured—that is, similar cells aggregate with each other, while dissimilar cells repel each other, ultimately forming a layered, rather than uniformly integrated structure. This invention, through specific cell ratios and preparation conditions, enables three types of homologous cells to co-aggregate into dense spheres with uniform structure and clear boundaries, thus avoiding cell sorting and the formation of uncertain structures such as "endothelial core + neural shell + DPSCs interlayer."

[0079] The present invention will now be described through the following embodiments.

[0080] Example 1: Construction of scaffold-free microspheres with prevascularization and preneuration dual functions derived from DPSCs

[0081] S1 obtains endothelial-like cells and neuron-like cells derived from DPSCs:

[0082] Rabbit third-generation DPSCs were induced for 14 consecutive days with endothelial induction medium (DMEM + 10% FBS + 10 μg / L bFGF + 0.1 mg / L Dex + 50 μg / L VEGF + 2 g / L BSA) to obtain endothelial-like cells.

[0083] Rabbit third-generation DPSCs were induced for 14 consecutive days with neural induction medium (neural basal A medium + 10% FBS + 40 ng / mL bFGF + 20 ng / mL EGF + 1×B27) to obtain neural-like cells.

[0084] S2 Construction of a multi-cell co-culture system: Undifferentiated DPSCs, endothelial-like cells, and neuron-like cells were digested, counted, and mixed at a cell ratio of 1:1:1 (total cell count 1×10⁻⁶). 5 Each well was seeded into an ultra-low adhesion 96-well plate, and 100 μL of a culture medium consisting of a mixture of DMEM complete medium, endothelial induction medium, and neural induction medium in proportion was added.

[0085] Culture and formation of S3 micro-tissue spheres: After 3 days of culture, scaffold-free micro-tissue spheres with dual functions of prevascularization and preneuration are formed.

[0086] Example 2:

[0087] Undifferentiated DPSCs, endothelial-like cells, and neuron-like cells were digested and counted, and mixed in a cell number ratio of 3:1:1, otherwise the same as in Example 1.

[0088] Example 3:

[0089] S1. Endothelial-like cells derived from DPSCs: Rabbit third-generation DPSCs were induced for 14 consecutive days with endothelial induction medium (DMEM + 10% FBS + 10 μg / L bFGF + 0.1 mg / L Dex + 50 μg / L VEGF + 2 g / L BSA) to obtain endothelial-like cells.

[0090] S2 Construction of a multi-cell co-culture system: Undifferentiated DPSCs were digested and counted with the above-mentioned endothelial-like cells, and mixed at a cell ratio of 1:1 (total cell count 1×10⁻⁶). 5 Each well was seeded into an ultra-low adhesion 96-well plate, and 100 μL of a culture medium consisting of an equal mixture of DMEM complete medium and endothelial induction medium was added.

[0091] Culture and formation of S3 micro-tissue spheres: After culturing in a 37℃, 5% CO2 incubator for 3 days, with half the medium replaced every other day, prevascularized cell micro-tissue spheres with a diameter of about 500μm can be formed.

[0092] Example 4:

[0093] Undifferentiated DPSCs were digested and counted with the above-mentioned endothelial-like cells, and mixed at a cell number ratio of 3:1, otherwise the same as in Example 3.

[0094] Example 5:

[0095] S1 Obtaining neuron-like cells derived from DPSCs: Rabbit third-generation DPSCs were induced for 14 consecutive days with neural induction medium (neural basal A medium + 10% FBS + 40 ng / mL bFGF + 20 ng / mL EGF + 1×B27) to obtain neuron-like cells.

[0096] S2 Construction of a multi-cell co-culture system: Undifferentiated DPSCs were digested and counted with the above-mentioned neuron-like cells, and mixed at a cell ratio of 1:1 (total cell number 1×10⁻⁶). 5 (100 μL of a mixture of DMEM complete medium and neural induction medium in equal proportions) were inoculated into 96-well ultra-low adhesion plates and added to the plate.

[0097] Culture and formation of S3 micro-tissue spheres: Dense pre-neuralized micro-tissue spheres were formed after 3 days of culture.

[0098] Example 6

[0099] The undifferentiated DPSCs were digested and counted with the above-mentioned neuron-like cells, and mixed at a cell number ratio of 3:1, otherwise the same as in Example 5.

[0100] Comparative Example 1

[0101] All undifferentiated DPSCs were inoculated and cultured to obtain tissue microspheres.

[0102] 1. Isolation, culture, and endothelial- and neural-oriented differentiation of primary rabbit DPSCs

[0103] 1.1 Experimental Procedure and Detection Methods:

[0104] (1) Isolation and culture of primary rabbit DPSCs: Rabbits were euthanized by rapid overdose injection of 2% lidocaine hydrochloride into the marginal ear vein. After disinfection of the maxillofacial region with povidone-iodine, the rabbit molars were extracted. The extracted molars were rinsed with PBS containing 2% penicillin and antibiotics and then placed in pre-cooled DMEM medium containing double the amount of penicillin and antibiotics for 20 min. The young rabbit molars were rinsed with PBS containing 2% penicillin and antibiotics. The intact pulp tissue was removed with a plucking needle and soaked in a six-well plate containing DMEM medium containing 3% penicillin and antibiotics. The pulp tissue was minced and 1 mL of 3 g / L type I collagenase was added. After mixing, the tissue was digested in a 37°C water bath. After 1 h, the digestion was stopped, and the supernatant was discarded by centrifugation. The tissue was resuspended in 2-3 mL of DMEM medium containing FBS and penicillin and antibiotics and inoculated into 25 mL culture flasks and cultured in a CO2 incubator. The culture medium was changed every 3 days, and the tissue was observed and photographed under an inverted microscope.

[0105] (2) Endothelial differentiation

[0106] Cell induction: Take 3rd generation DPSCs at 5×10⁻⁶ 5 Cells were seeded per well in 6-well plates. When the cells reached 90% confluence, endothelial cell induction medium (specific ratio: DMEM + 10% FBS + 1% penicillin-dextrin antibiotics + 10 μg / L basic fibroblast growth factor + 0.1 mg / L dexamethasone + 50 μg / L vascular endothelial growth factor + 2 g / L bovine serum albumin) was added, and the medium was changed every 3 days. On day 14 of induction, the morphology of the induced cells was observed and photographed under an inverted microscope.

[0107] Matrigel tube formation assay: Matrigel, pipettes, pipette tips, and 96-well plates were pre-chilled at 4°C. In a clean bench, the 96-well plates were placed on ice, and 50 μl of Matrigel was slowly added to each well before incubation for 30 min. DPSCs and endothelial-induced endothelial-like cells were digested and centrifuged, then resuspended in complete culture medium and endothelial cell induction medium, respectively. After removing the 96-well plates from the incubator, the tubes were loaded onto the plates at 4 × 10⁶ cells / well. 4 DPSCs and endothelial-like cells were seeded at a density of cells / well, respectively, and cultured in an incubator. Tube formation was observed under an inverted microscope after 4 hours, and photographs were taken. The percentage of vessel area in both groups was calculated using AngioTool software.

[0108] The relative expression level of the angiogenesis-related gene VEGF was detected by qRT-PCR: Two groups of cells (endothelial cells induced by endothelial transformation in the experimental group and DPSCs in the control group) were digested with trypsin and centrifuged. After cell collection, 0.5 mL of Buffer RL lysis buffer was added to each group to extract total RNA. The purity and concentration of the RNA samples were determined and stored at -80℃. RNA reverse transcription was performed to obtain the corresponding cDNA, which was stored at -20℃. PCR amplification primers were designed based on the rabbit VEGF gene sequence, with GAPDH as an internal control. The gene sequence is shown in Table 1. qRT-PCR amplification was performed using a PCR instrument (iQ5, Bio-Rad, USA). The relative expression level of the target gene mRNA was calculated according to the following formula.

[0109] Y = 2 - (∆∆Ct)

[0110] Y represents the relative mRNA expression level of VEGF; △△Ct represents the ratio of VEGF Ct value to GAPDH Ct value between endothelial-like cells and DPSCs (VEGF Ct value to GAPDH Ct value).

[0111] Table 1. Gene sequence of VEGF, an angiogenesis marker gene

[0112]

[0113] (3) Neural differentiation

[0114] Cell induction: Take 3rd generation DPSCs at 5×10⁻⁶ 5 Cells were seeded per well in 6-well plates. When the cells reached 90% confluence, the original culture medium was removed, and neural cell induction medium (specific ratio: neural basal A medium + 10% FBS + 1% antibiotics + 40 ng / mL basic fibroblast growth factor + 20 ng / mL epidermal growth factor (EGF) + 1×B27) was added). The medium was changed every 3 days. On day 14 of induction, cell morphology and structure were observed under a microscope.

[0115] Giemsa staining: After 14 days of neuralization induction, DPSCs were stained with Giemsa stain to assess cell differentiation. The culture medium was discarded, and the cells were washed three times with PBS. Giemsa staining solution was added to six-well plates, and the plates were incubated for 0.5 h. The staining solution was aspirated, and the plates were gently rinsed with running water. After observing under a microscope that no excess dye was observed and all cells were stained blue-purple, the neuralization of the cells was observed under an inverted microscope, and photographs were taken.

[0116] The relative expression level of the neurogenesis-related gene NCAM was detected by qRT-PCR: PCR amplification primers were designed based on the rabbit NCAM gene sequence, with GAPDH as an internal control. The gene sequence is shown in Table 2. The relative expression level of the target gene mRNA was calculated according to the following formula.

[0117] Y = 2 - (∆∆Ct)

[0118] Y represents the relative mRNA expression level of NCAM; △△Ct represents the ratio of endothelial-like cells (NCAM Ct value - GAPDH Ct value) to DPSCs (NCAM Ct value - GAPDH Ct value).

[0119] Table 2. Gene sequences of NCAM

[0120]

[0121] 1.2 Results

[0122] like Figure 1-7 As shown, after 3 days of conventional culture, cells can be seen "crawling out" of the tissue block. Figure 1 A) Cells grow outwards in a spiral or radial pattern from the center of the tissue block, proliferating rapidly. Most cells resemble fibroblasts in morphology, appearing spindle-shaped or irregularly triangular. After passage, the cell proliferation rate is faster than in the primary culture. Figure 1 B), after approximately 3-4 days, it can cover 80%-90% of the bottom area of ​​the vial. DPSCs, after 14 days of endothelial induction ( Figure 2 Endothelial-like cells are spindle-shaped or polygonal, with relatively tight intercellular connections, arranged in a "paving stone" pattern. Matrigel tubule formation experiments showed ( Figure 3 Both DPSCs and endothelial-like cells can form interconnected vascular-like structures, with endothelial-like cells forming significantly more vascular-like structures than DPSCs. Quantitative results showed ( Figure 4 The percentage of vessel area in the angiogenic structures formed by endothelial-like cells was 77.1±1.30%, while the percentage of vessel area in the angiogenic structures formed by DPSCs was only 18.97±2.88%. Endothelial-like cells showed a significantly higher percentage than DPSCs (P<0.001), indicating that endothelial-like cells induced by endothelialization for 14 days possess strong angiogenic capacity. qRT-PCR results showed ( Figure 5 The relative expression level of VEGF in endothelial-like cells obtained after 14 days of endothelial induction from DPSCs was significantly higher than that in DPSCs, indicating that DPSCs have endothelial differentiation capacity and the induced endothelial-like cells have strong angiogenic ability. Giemsa staining results of neuron-like cells obtained after 14 days of neural induction from DPSCs showed ( Figure 6The induced differentiated cells exhibited typical neuron-like morphology, with clearly visible and slender neurites, indicating that DPSCs possess neurogenic differentiation potential and could be successfully induced to differentiate into neuron-like cells. qRT-PCR results showed ( Figure 7 After 14 days of neural induction, the relative expression level of NCAM in neuron-like cells was significantly higher than that in DPSCs, indicating that DPSCs have neurogenic differentiation potential and the induced neuron-like cells have strong neurogenic ability.

[0123] 2. Construction and characterization of micro-tissue spheres

[0124] 2.1 Experimental Procedure and Detection Methods

[0125] Seven groups of cell microspheres from Examples 1-6 and Comparative Example 1 were cultured for 3 days and then removed (n=6), fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Subsequently, histological staining of the cell microspheres was performed.

[0126] HE staining: After dewaxing, the sections were immersed in hematoxylin staining solution for 5 min, rinsed with running tap water to remove residual stain, differentiated with 0.7% hydrochloric acid ethanol for 10 s, rinsed with running water again, stained with eosin solution for 30 s, then dehydrated and cleared, and successively immersed in 95% ethanol I for 30 s, 95% ethanol II for 30 s, anhydrous ethanol I for 30 s, anhydrous ethanol II for 30 s, xylene I for 30 s, and xylene II for 30 s. After drying, neutral resin was added for mounting, and the sections were observed and photographed under an inverted microscope.

[0127] Masson staining: After dewaxing, smear the sections with iron hematoxylin for 15 min, rinse with running water, stain with Ponceau S for 5-10 min, rinse with running water, treat with phosphomolybdic acid for 5 min, then stain with aniline blue for 5 min, rinse with running water. Treat with glacial acetic acid for 1 min, rinse with running water, dehydrate, fix, mount, and air dry. Observe and photograph under an inverted microscope.

[0128] 2.2 Results

[0129] like Figure 8-10 As shown, using the above method, seven groups of micro-tissue spheres with different cell ratios and combinations were successfully constructed (including DPSCs alone, DPSCs / endothelial-like cells, DPSCs / neuronal-like cells, and a mixture of the three). Gross observation and microscopic examination showed that the cells in all groups could rapidly aggregate within 24 hours and form dense spheres with clear boundaries and a diameter of approximately 400-600 μm after 3 days. Figure 8 A). During aspiration and transfer using a pipette, the micro-tissue spheres maintained their morphology intact, without loosening or deformation, indicating excellent mechanical integrity and the ability to withstand transplantation procedures. Figure 8B). HE staining results showed ( Figure 9 Cells and extracellular matrix constitute DPSC-derived cell microspheres. These microspheres are round or oval in shape, with relatively tightly packed cells on the outside and a looser arrangement inside. The cells are flattened and elongated, and the seventh group's microspheres are more densely packed than the others, indicating that all groups of cell microspheres aggregated relatively tightly. The seventh group (all constructed from DPSCs) showed the most compact aggregation, demonstrating that DPSCs possess complete self-assembly capabilities. Masson staining results showed ( Figure 10 In all seven groups of cell microspheres, a large amount of blue collagen fibers were generated between cells, indicating that the cell microspheres possess a certain degree of toughness and tensile strength. The experiment also showed that when the cell ratio exceeded the limits specified in this invention, the microsphere structure became loose or the level of prefunctionalization significantly decreased, confirming the crucial importance of the ratio selection in this invention.

[0130] 3. In vitro verification of prevascularization and preneurization functions

[0131] 3.1 Experimental Procedure and Detection Methods:

[0132] After culturing the seven groups of cell microspheres for 3 days (n=6), they were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Subsequently, histological staining of the cell microspheres was performed.

[0133] Immunofluorescence staining: The tissue sections were placed in citrate antigen retrieval solution, autoclaved, and then placed in a 100℃ oven for 45 min. After natural cooling, they were rinsed three times with PBS for 3 min each time. Blocking was performed with 3% hydrogen peroxide for 10 min, followed by rinsing three times with PBS. Primary antibody incubation: Incubation was performed overnight in a humidified chamber at 4℃. The CD31 primary antibody was diluted 1:100, and the sections were washed three times with PBS for 3 min each time. Secondary antibody incubation: Fluorescent secondary antibody was added and incubated in the dark for 15 min, followed by rinsing three times with PBS for 3 min each time. Mounting: The sections were mounted with anti-quenched fluorescent mounting medium (containing DAPI), and observed and photographed under a microscope. The average fluorescence intensity of the angiogenic specific marker CD31 was analyzed using ImageJ image analysis software.

[0134] Immunohistochemical staining (ABC kit method): After dewaxing the above tissue sections, antigen retrieval was performed for 20 min, followed by rinsing twice with PBS for 3 min each time. After adding 3% hydrogen peroxide, the sections were incubated at 37°C for 20 min, followed by rinsing twice with PBS. Goat serum was used for blocking in the incubator for 30 min, then the serum was discarded, and the primary antibody (Nestin) was added directly without rinsing. The sections were placed overnight at 4°C. The next day, the sections were removed and placed at room temperature for 30 min, rinsed twice with PBS, and incubated with secondary antibody for 30 min. Simultaneously, solutions A and B and PBS were mixed at a ratio of 10 μL:10 μL:1 mL, and allowed to stand at room temperature for 30 min. After secondary antibody incubation, the sections were washed twice with PBS, and the pre-prepared mixture was added and incubated for 30 min. After washing twice with PBS, the chromogenic solution (solution A:solution B = 1:50) was added. Once the tissue showed a brown color, the chromogenic solution was discarded, and the sections were rinsed three times with running water. The sections were then immersed in hematoxylin for 40 s, and the staining was observed under an inverted microscope. After rinsing with running water, the slides were dried, fixed, and mounted. They were then observed and photographed. The percentage of Nestin-positive cells, a neuroblast-specific marker, was analyzed using ImageJ image analysis software.

[0135] 3.2 Results:

[0136] like Figure 11-14 As shown, angiogenesis potential was assessed using immunofluorescence staining to detect the vascular endothelial marker CD31. Results showed that all groups containing endothelioid cells (Examples 1-4) were strongly positive for CD31, while groups without endothelioid cells (Examples 5 and 6, Comparative Example 1) showed no positive expression. This demonstrates that CD31 expression is entirely dependent on the presence of endothelioid cells—positive in the presence of endothelioid cells and negative in their absence, ruling out the possibility that DPSCs spontaneously express mature endothelial markers during the in vitro stage (3 days of culture). Among these, the groups consisting solely of DPSCs and endothelioid cells (Examples 3 and 4) exhibited the highest fluorescence intensity. The fluorescence intensity of groups containing all three cell types (Examples 1 and 2) was lower than that of Examples 3 and 4, indicating that endothelioid cells possess the strongest pre-angiogenic potential, are the sole source of CD31 signaling, and this signal is not interfered with by the presence of neuron-like cells. Furthermore, the ratio of DPSCs to endothelial cells affects the final endothelial maturity.

[0137] Neurogenic Potential: Immunohistochemical staining was used to detect the neural stem cell marker Nestin. Results showed that all groups containing neuron-like cells (Examples 1, 2, 5, and 6) exhibited brownish-red positive signals, while groups without neuron-like cells (Examples 3-4 and Comparative Example 1) showed no positive expression. This demonstrates that Nestin expression is entirely dependent on the incorporation of neuron-like cells; groups consisting only of DPSCs or DPSCs + endothelial-like cells (Examples 3, 4, and Comparative Example 3) failed to spontaneously form neural progenitor cells after 3 days of in vitro culture, ruling out the possibility of DPSCs spontaneously differentiating into the neural lineage without induction; among these, the groups composed solely of DPSCs and neuron-like cells (Examples 5 and 6) showed the highest percentage of positive area, exhibiting the strongest preneurogenic potential.

[0138] 4. Cell viability assessment

[0139] 4.1 Experimental Procedure and Detection Methods:

[0140] After 3 days of culture, cell viability of the microspheres was assessed using a live / dead cell staining kit. After removing the microspheres, they were washed twice with PBS to remove residual culture medium. First, prepared propidium iodide (PI) working solution was added, ensuring the microspheres were completely submerged, and incubated at room temperature for 10 min. The PI working solution was removed, and the cells were gently washed once with PBS to remove the supernatant. Then, prepared calcein working solution was added, ensuring the cells were completely submerged, and incubated at room temperature for 45 min. The calcein working solution was removed, and the cells were gently washed once with PBS to remove the supernatant. PBS was added, and the cell live / dead cell labeling was observed and photographed under a confocal microscope. Dead cells exhibiting red fluorescence were quantified using ImageJ image analysis software.

[0141] 4.2 Results.

[0142] like Figure 15 , 16 As shown, double staining of live / dead cells indicated high internal viability in all seven groups of micro-tissue spheres. However, compared to micro-tissue spheres composed entirely of undifferentiated DPSCs (Comparative Example 1), micro-tissue spheres incorporating endothelial-like cells and / or neuron-like cells (Examples 1-6) exhibited a significantly reduced number of dead cells in their central region. This demonstrates that predifferentiated cells significantly improve material exchange in the core region of the three-dimensional structure by enhancing intercellular interactions or regulating the microenvironment, thereby increasing overall cell viability.

[0143] 5. Verification of in vivo pulp regeneration effect

[0144] To simulate clinical pulp regeneration, seven groups of micro-tissue balls were implanted into the root canals of treated rabbit molars and transplanted subcutaneously into nude mice for 8 weeks to evaluate ectopic pulp regeneration.

[0145] 5.1 Testing Method:

[0146] (1) General observation

[0147] The survival status of nude mice and whether the implant on their backs has fallen off were observed regularly each week. During the sampling process, the relationship between the subcutaneous tissue and the tooth roots was observed, and the condition of the tooth roots was also observed.

[0148] (2) Histological staining

[0149] The root samples from each group were fixed with 4% paraformaldehyde, decalcified with EDTA, and then subjected to HE, Masson staining, and immunohistochemical staining (CD31, GFAP, Nestin) to observe the regeneration of dental pulp tissue.

[0150] (3) Microvessel counting

[0151] Three HE-stained sections were selected for each group. First, densely vascularized areas were selected under a 100x microscope. Then, the number of microvessels was counted in three fields of view for each section under a 200x microscope. Hemorrhage areas, fibrosis, and edge reaction areas were excluded. The average of the three counts was taken as the number of microvessels in the group.

[0152] 5.2 Results:

[0153] like Figure 17-27 As shown.

[0154] Figure 17 This refers to the surgical procedure of subcutaneous heterotopic transplantation of DPSC-derived cell microtissue spheres in nude mice. Figure 18 This study presents a gross observation of seven groups of DPSC-derived cell microspheres and a blank control group (filled with calcium hydroxide) after ectopic transplantation. As shown in the figure, tissue samples were taken 8 weeks after implantation, revealing that all teeth were surrounded by subcutaneous fibrous connective tissue from nude mice. Except for the blank control group, red pulp-like new tissue was observed through the dentin in all groups, indicating angiogenesis. In the blank control group, yellow calcium hydroxide solvent was visible in the root canal of rabbit molars, with no new tissue formation, indicating that the microspheres not only survived after implantation but also established a functional connection with the host circulatory system (blood perfusion caused the new tissue to appear red). This phenomenon was not observed in the blank control group, ruling out interference from residual blood in the tooth root itself.

[0155] HE staining results of seven groups of DPSC-derived cell microspheres and a blank control group after heterotopic transplantation showed that ( Figure 19In all seven groups of root canals filled with DPSC-derived cell micro-tissue spheres, regenerated tissue essentially filled the entire root canal. Numerous tubular vascular-like structures were observed within the regenerated tissue in each group, containing anucleated erythrocytes. The blank control group showed no formation of any new vascular-like structures, indicating successful reconstruction of the functional vascular network. The "tubular vascular-like structures" indicate that these are not scattered endothelial cells, but rather mature blood vessels with lumens. The presence of "anucleated erythrocytes" suggests that these tubular structures are perfused with blood and are true functional blood vessels, not merely immature endothelial strands expressing CD31. This demonstrates that the seven groups of rabbit molars filled with DPSC-derived cell micro-tissue spheres formed vascularized pulp tissue in nude mice, suggesting that the seven groups of DPSC-derived cell micro-tissue spheres have strong pulp tissue regeneration capacity and the potential to reconstruct the pulp vascular network, achieving excellent ectopic pulp regeneration results.

[0156] Microvessel counting results showed ( Figure 20 All seven groups of cell micro-tissue spheres showed microvascular formation after ectopic transplantation. Examples 3 and 4 showed a relatively high number of newly formed microvessels, while Comparative Example 1 showed the fewest. Furthermore, the number of newly formed blood vessels in Examples 3 and 4 was significantly higher than that in Comparative Example 1, with statistically significant differences. No statistically significant differences were found between any of the other groups. This indicates that all seven groups of cell micro-tissue spheres have the ability to form blood vessels and construct vascular networks, and Examples 3 and 4 showed a significantly higher ability to form blood vessels than Examples 3 and 4.

[0157] Masson's results after heterotopic transplantation of seven groups of DPSC-derived cell microspheres and a blank control group showed that ( Figure 21 Except for the blank control group, blue collagen fiber-like structures were observed inside the root canals of rabbit molars after ectopic transplantation of cell microspheres in all groups, mostly clustered in the apical region; vascular-like structures containing erythrocytes were observed in the mesenchymal tissue of the pulp center. In Examples 5, 6, and Comparative Example 1, obvious local necrotic foci were observed near the crown. In Examples 1, 5, 6, and Comparative Example 1, odontoblasts were observed arranged in a palisade pattern, indicating that the regenerated pulp tissue of Examples 1, 5, 6, and Comparative Example 1 has the potential to form dentin.

[0158] Figures 22 to 27The figures show immunohistochemical staining images of CD31, Nestin, and GFAP after ectopic transplantation of seven groups of DPSC-derived cell micro-tissue spheres and a blank control group, as well as bar charts showing the percentage of CD31, Nestin, and GFAP positive cells. As shown, all seven groups of regenerated tissues showed positive expression of CD31, and the percentage of positive area in Examples 3 and 4 was higher than in the other groups, but the differences between groups were not statistically significant. The newly formed tissues of the seven implants showed positive expression of Nestin and GFAP, specific markers related to nerve formation, and the percentage of positive area in Examples 5 and 6 was higher than in the other groups, but the differences were not statistically significant. This indicates that the rabbit molars filled with cell micro-tissue spheres formed vascularized and neuralized pulp-like tissue in nude mice, suggesting that the seven groups of cell micro-tissue spheres have pulp regeneration potential. Even in the groups without added nerve-like cells in vitro (Examples 3, 4, and Comparative Example 1), Nestin and GFAP positive structures were detected in vivo 8 weeks after implantation. This indicates that angiogenesis in newly formed tissues can recruit endogenous neural progenitor cells from the host or induce DPSCs to differentiate into neural lineages in vivo, forming a synergistic regenerative microenvironment.

[0159] Examples 1-2 simultaneously expressed CD31 and Nestin in vitro, and after in vivo transplantation, they showed the best synergistic vascularization and neuralization of dental pulp regeneration. The positive areas of blood vessels and nerves were significantly higher than those of other groups, the vascularization and neuralization process was faster, and the cell survival rate in the central region was significantly improved compared with pure DPSCs microspheres, realizing dental pulp tissue regeneration with both structural and functional biomimicry.

[0160] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the invention. Those skilled in the art will understand that implementing all or part of the above-described embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A scaffold-free multicellular microtissue sphere for dental pulp regeneration, characterized in that, The scaffold-free multicellular micro-tissue spheres are dense spheres formed spontaneously by dental pulp stem cells, endothelial-like cells and / or neuron-like cells through intercellular interactions. The endothelial-like cells and neuron-like cells are obtained by induced differentiation of dental pulp stem cells.

2. The scaffold-free multicellular micro-tissue sphere for pulp regeneration as described in claim 1, characterized in that, The ratio of the number of dental pulp stem cells, endothelial-like cells, and neuron-like cells is 2-10:0-5:0-5, and the number of endothelial-like cells and neuron-like cells is not 0 at the same time.

3. The scaffold-free multicellular microtissue sphere for pulp regeneration as described in claim 2, characterized in that, The ratio of dental pulp stem cells, endothelial-like cells, and neuron-like cells is one of 1:1:1, 3:1:1, 1:1:0, 3:1:0, 1:0:1, or 3:0:

1.

4. A method for preparing scaffold-free multicellular microspheres for pulp regeneration as described in claim 1, characterized in that, Includes the following steps: S1 Obtaining endothelial-like cells and / or neuron-like cells derived from DPSCs: Dental pulp stem cells were cultured in endothelial cell induction medium and / or neuron cell induction medium, respectively, and induced to differentiate into endothelial-like cells and / or neuron-like cells, respectively, to obtain endothelial-like cells and / or neuron-like cells. S2 constructs a multi-cell co-culture system: DPSCs, endothelial-like cells and / or neuron-like cells are mixed and seeded in a low-adhesion culture plate; Culture and formation of S3 micro-tissue spheres: The cells were continuously cultured in an incubator at 37℃ and 5% CO2. During this period, the cells spontaneously aggregated and formed spherical, scaffold-free, three-dimensional multicellular micro-tissue spheres.

5. The method for preparing scaffold-free multicellular microspheres for pulp regeneration as described in claim 4, characterized in that, The S1 method yields endothelial-like cells and neuron-like cells derived from DPSCs, which need to be cultured for 10-14 days.

6. The method for preparing scaffold-free multicellular microspheres for pulp regeneration as described in claim 4, characterized in that, The S1 method obtains endothelial-like cells and nerve-like cells derived from DPSCs. The endothelial cell induction culture medium is a basal culture medium supplemented with basic fibroblast growth factor, dexamethasone, vascular endothelial growth factor, and bovine serum albumin.

7. The method for preparing scaffold-free multicellular microspheres for pulp regeneration as described in claim 4, characterized in that, The nerve cell induction culture medium is a basic nerve culture medium supplemented with basic fibroblast growth factor, epidermal growth factor and B27 additive.

8. The method for preparing scaffold-free multicellular microspheres for pulp regeneration as described in claim 4, characterized in that, In S3, the culture is carried out in an incubator for more than 3 days.

9. The application of the scaffold-free multicellular microspheres for pulp regeneration as described in claim 1 in pulp tissue regeneration grafts to promote vascularization and / or neuralization of pulp tissue regeneration.

10. The application of the scaffold-free multicellular micro-tissue spheres for pulp regeneration as described in claim 9 in pulp tissue regeneration grafts, characterized in that, The graft is used to implant into the pulp chamber or root canal after pulp removal.

Images