CN115054731A

CN115054731A - Injectable functional heterogeneous microsphere and preparation method and application thereof

Info

Publication number: CN115054731A
Application number: CN202210424372.1A
Authority: CN
Inventors: 谢志坚; 史洋; 张淳; 王刚; 谢明君; 贺永; 石珏; 陈卓; 陆炜英; 宓锐
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2022-09-16
Anticipated expiration: 2042-04-21
Also published as: CN115054731B

Abstract

The invention discloses an injectable functionalized heterogeneous microsphere and a preparation method and application thereof, belonging to the field of biomedical tissue engineering. The preparation method comprises the steps of preparing microsphere phase and pipeline phase electric spraying ink, extracting dental pulp mesenchymal stem cells of a patient, inducing differentiation of blood vessels, obtaining cell-carrying pipeline phase electric spraying ink, preparing heterogeneous microspheres, solidifying, separating, washing and functionalizing. The invention realizes the double-unit integrated preparation of the functionalized micro-pipeline structure and the microsphere structure through a coaxial electric spraying manufacturing process, provides favorable conditions for the rapid vascularization in clinical ascending large-area bone augmentation surgery, greatly simplifies the operation due to the injectability and provides a new idea for the research, development and application of the functionalized tissue engineering microsphere.

Description

Injectable functional heterogeneous microsphere and preparation method and application thereof

Technical Field

The invention belongs to the field of tissue engineering, and particularly relates to a preparation method and application of an injectable functional microsphere for promoting rapid vascularization and early osteogenesis.

Background

Jaw trauma, postoperative tumor, infection and the like often cause large-area defect of oral and maxillofacial bone tissues, the incidence rate of the defect accounts for about 40-50% of oral and maxillofacial diseases, the defect not only affects the chewing function, the attractiveness, the pronunciation and the like of a patient, but also causes certain damage to the psychology and the general condition of the patient to different degrees, and the life quality of the patient is greatly reduced. Generally, bone tissue has a unique self-regenerating and healing capacity, but when a bone defect exceeds a limit (>5-8mm), its self-healing capacity is often not ideal due to a lack of timely blood supply in the central portion of the defect. Clinically, oral bone tissue defects, particularly insufficient amounts of alveolar bone over a wide range, directly affect the oral repair treatment of patients, i.e., the reconstruction of chewing function and the restoration of aesthetic appearance. In particular, the artificial tooth implantation has become the most popular treatment method in the current oral cavity repair, and the requirement for alveolar bone mass is quite high, and if the bone mass is insufficient, the difficulty of the implant operation is greatly increased, and even the artificial tooth implantation repair cannot be carried out. Although autologous bone grafting is the gold standard for bone reconstruction in large-area bone defect in clinic, a second operation area needs to be opened up, complications such as pain and nerve injury in different degrees can be caused, and the autologous bone grafting material cannot be accepted by most patients, so that the autologous bone grafting material is widely applied to clinic. When the size of the graft material is more than 200 mu m, the diffusion limit of nutrition and oxygen can be exceeded, a nutrition network needs to be constructed to avoid the problem of unsmooth internal nutrition transportation, but the current bone graft material does not have the functions of a nutrition flow pipeline and an induced angiogenesis. In addition, because the rapid vascularization can not only ensure the oxygen and nutrition exchange of the central part, but also bring osteogenic precursor cells and provide necessary conditions for the early formation of new bones, the vascularization inducing factors and/or endothelial cells are introduced into the stent, so that the rapid vascularization of the large bone graft, especially the central part far away from the receiving area, can be realized, the early bone reconstruction is further realized, and favorable conditions are provided for the early implantation repair and the restoration of the beauty and the function of patients. In particular, for the maxillary posterior tooth loss, the maxillary sinus floor lift is a more commonly used bone augmentation method, a bone graft material is often implanted at a position between a cortical bone of the maxillary sinus floor and a maxillary sinus membrane, but this region often lacks abundant blood supply, so that a time required for inducing formation of a new bone is long, it is often necessary to discharge energy to implant an implant for half a year, and further, a central position far from the sinus membrane and a bone wall is difficult to regenerate the bone due to lack of timely blood supply and osteogenesis precursor cells, and particularly when inflammation is present in the maxillary sinus, this region is easily infected and thus operation fails.

In addition, the bone graft which is most commonly used clinically at present is in the form of granular bone powder, the operability is poor, a small curette is often required to be repeatedly taken and placed for many times to fill the whole bone defect, and a large amount of operation time is required for filling the bone powder for a large-area bone defect. In particular, in the maxillary sinus floor lifting operation, since the maxillary sinus is located in the upper jaw, the small granular bone powder is easy to drop down after being implanted due to the action of gravity, so that the maxillary sinus floor lifting operation needs to be repeatedly implanted. Moreover, the existing bone graft particles are small and have no viscoelasticity, and are easy to fall off or adhere to soft tissues in the non-operative area of the oral cavity, so that the operation process is delayed. Therefore, the injectable vascularized osteogenic microsphere scaffold can provide convenience for large-area bone augmentation surgery, especially maxillary sinus floor elevation surgery on the upper jaw.

Disclosure of Invention

The invention aims to overcome the defects in bone grafting materials in the prior art, and provides an injectable functionalized heterogeneous microsphere and a preparation method and application thereof. The invention is inspired by the famous fluid phenomenon of the rope rolling effect, and realizes the integrated preparation of a pipeline unit and a microsphere unit by introducing the stainless steel coaxial double-nozzle into an electric spraying device, thereby providing technical support for constructing the multifunctional heterogeneous microsphere. Meanwhile, vascularization induced hDPSC and vascular endothelial growth factor are loaded in the pipeline ink, and bone morphogenetic protein is loaded in the microsphere ink, so that the vascularization and osteogenesis promotion dual functions of the heterogeneous microspheres are realized. And finally, the functionalized heterogeneous microspheres are injected to a bone defect area through the injectable characteristic of the gel microspheres, so that the aims of rapid vascularization and early osteogenesis are fulfilled, and favorable conditions are provided for early planting and repairing.

The invention adopts the following specific technical scheme:

in a first aspect, the invention provides a preparation method of an injectable functionalized heterogeneous microsphere, which comprises the following steps:

s1: sequentially dissolving a photoinitiator and bone morphogenetic protein in a methacrylic acid acylated gelatin solution to obtain a first mixed solution; then adjusting the pH of the obtained first mixed solution to be neutral, and filtering the first mixed solution through a 0.22 mu m filter to obtain sterile microsphere phase electronic injection ink;

s2: sequentially dissolving a photoinitiator and vascular endothelial growth factors in a medical gelatin solution to obtain a second mixed solution; then adjusting the pH value of the obtained second mixed solution to be neutral, and filtering the second mixed solution by a 0.22 mu m filter to obtain sterile pipeline phase electric spraying ink;

s3: extracting a target dental pulp mesenchymal stem cell for parallel functional induction to obtain an vascularization induced dental pulp mesenchymal stem cell;

s4: resuspending the obtained vascularization induced dental pulp mesenchymal stem cells by using the sterile pipeline electric phase jet water to obtain cell-loaded pipeline electric phase jet ink;

s5: continuously forming a plurality of micro-droplets at the ambient temperature of 37 ℃ and in a high-voltage electric field by using an electric spraying device provided with coaxial double nozzles based on the obtained cell-carrying pipeline phase electric jet ink and the sterile microsphere phase electric jet ink, and simultaneously pre-curing the obtained micro-droplets to obtain pre-cured micro-droplets; the obtained precured micro-droplets continuously fall into a main receiving pool filled with the dimethyl silicone oil, and the precured micro-droplets in the main receiving pool are continuously stirred by a stirring device; finally, performing final solidification on the precured micro-droplets in the main receiving pool to obtain first heterogeneous microspheres;

the coaxial double nozzles comprise a first nozzle and a second nozzle which are coaxially sleeved inside and outside, the first nozzle positioned at the outside is used for supplying sterile microsphere phase electric spraying ink, the second nozzle positioned at the inside is used for supplying cell-carrying pipeline phase electric spraying ink, so that the outside of the obtained micro liquid drop is of a microsphere structure formed by the sterile microsphere phase electric spraying ink, and the inside of the obtained micro liquid drop is of a micro pipeline structure formed by the cell-carrying pipeline phase electric spraying ink;

s6: the obtained first heterogeneous microspheres and the simethicone are together transferred to a cold storage and solidification treatment at 4 ℃, and then the simethicone and residual charges on the first heterogeneous microspheres are removed to obtain second heterogeneous microspheres;

s7: and putting the obtained second heterogeneous microsphere into an angiogenesis induction culture medium for culturing for 3-7 days to dissolve medical gelatin, so that a micro-pipeline beneficial to the adhesion and proliferation of the dental pulp mesenchymal stem cells is formed, and the injectable functional heterogeneous microsphere is obtained.

Preferably, the solution of the methacrylic acylated gelatin is obtained by dissolving the methacrylic acylated gelatin with physiological saline under aseptic conditions; the medical gelatin solution is obtained by dissolving medical gelatin in physiological saline under aseptic conditions.

Preferably, in the first mixed solution, the substitution rate of the methacrylated gelatin is 30%, the mass concentration is 5%, the mass concentration of the photoinitiator is 0.5%, and the concentration of the bone morphogenic protein is 600 to 800ng/mL (preferably 600 ng/mL).

Preferably, the photoinitiator is a blue light initiator, more preferably LAP or L2959.

Preferably, the second mixed solution contains medical gelatin 15 wt%, photoinitiator 0.5 wt%, and vascular endothelial growth factor 200-400 ng/mL (preferably 200 ng/mL).

Preferably, the density of the dental pulp mesenchymal stem cells in the cell-carrying pipeline phase electric ink-jet water can be adjusted to 10-15 ten thousand/ml.

Preferably, the coaxial dual spray head is assembled by inserting a 27G stainless steel spray head into a 16G stainless steel spray head.

Preferably, the flow rate of the first spray head is 50-150 muL/min, the flow rate of the second spray head is 2-20 muL/min, and the voltage of the high-voltage electric field is 2.0-4.6 kV.

In a second aspect, the present invention provides an injectable functionalized heterogeneous microsphere obtained according to any one of the preparation methods of the first aspect.

In a third aspect, the present invention provides the use of an injectable functionalized heterogeneous microsphere according to the second aspect in the preparation of a bone remodeling medicament for promoting vascularization and early bone formation.

Compared with the prior art, the invention has the following beneficial effects:

1) the invention realizes the integrated preparation of the double units of the 'pipeline' and the 'microsphere' by utilizing the coaxial double-nozzle electric spraying process, so that the bracket contains a microvascular structure, the complex operation process of inoculating cells after the bracket is prepared is not needed, the problem that the central part of a large-area bone defect implanted with a bone grafting material cannot be vascularized in time is solved, the early vascularization of the grafting material is promoted while the central area is prevented from ischemic necrosis, and the early formation of new bones is induced.

2) The invention utilizes the injectability of the gel microspheres to assemble the heterogeneous microspheres into the injector, and the heterogeneous microspheres are conveyed to an operation area through the injector to quickly fill large-area bone defects, particularly when the maxillary sinus floor lifting operation is performed, the functionalized heterogeneous microspheres can be conveyed into the sinus cavity in one step through an injection mode, so that the bone meal is prevented from being taken and placed for many times, the operation time is shortened, and the heterogeneous microspheres have certain viscoelasticity, have certain protection effect on a maxillary sinus membrane, and can reduce the possibility of sinus membrane perforation.

3) The invention utilizes dental pulp mesenchymal stem cells of the wisdom teeth or non-functional teeth of a patient to carry out directional induced differentiation, and prints the cells into microspheres through an integrated electronic injection process to obtain tissue engineering heterogeneous microspheres, on one hand, the invention can quickly establish blood supply in vivo and induce the early formation of new bones, on the other hand, the invention avoids the immunogenicity problem while changing autologous tissues into valuables, and is beneficial to clinical transformation.

Drawings

FIG. 1 is a schematic flow diagram of a manufacturing process of the present invention;

FIG. 2 is a schematic structural diagram (A) and a representation diagram (B) of an injectable functionalized heterogeneous microsphere obtained by the invention;

FIG. 3 is a graph of the rheological properties of an aseptic tube phase electrospray ink and an aseptic microsphere phase electrospray ink;

fig. 4 is a schematic diagram illustrating a functionalized heterogeneous microsphere of a vascularly-induced human dental pulp mesenchymal stem cell and a clinical scenario thereof.

Detailed Description

The invention will be further elucidated and described with reference to the drawings and the detailed description. The technical features of the embodiments of the present invention can be combined correspondingly without mutual conflict.

As shown in fig. 1, the preparation method of an injectable functionalized heterogeneous microsphere provided by the present invention comprises the following specific steps:

1) preparing sterile microsphere phase electronic injection ink:

GelMA (i.e., methacrylated gelatin) was dissolved in physiological saline under aseptic conditions to obtain a GelMA solution. And then, uniformly dissolving the photoinitiator and the bone morphogenetic protein in the GelMA solution in sequence to obtain a first mixed solution. Since the first mixed solution is weakly acidic, the pH of the solution is adjusted to neutral (7.4 in this example) by adding a suitable amount of sodium hydroxide, and the solution is filtered through a 0.22 μm filter to obtain the sterile microsphere phase electrospray ink.

In this example, the substitution rate of GelMA was 30%, the mass concentration was 5%, the mass concentration of the photoinitiator was 0.5%, and the concentration of bone morphogenic protein was 600ng/mL in the first mixed solution. The photoinitiator may be blue light initiator LAP or L2959, the LAP used in this example.

2) Preparing sterile pipeline phase electronic spraying ink:

dissolving medical gelatin with physiological saline under aseptic condition to obtain medical gelatin solution. And then, uniformly dissolving the photoinitiator and the vascular endothelial growth factor in the medical gelatin solution in sequence to obtain a second mixed solution. And then adjusting the pH value of the second mixed solution to be neutral, and filtering the second mixed solution through a 0.22 mu m filter to obtain the sterile pipeline phase electrospray ink.

In this embodiment, in order to form a more obvious "rope winding" phenomenon in the internal and external two-phase electrical spraying ink subsequently flowing through the coaxial dual-nozzle, more vascularization induced dental pulp mesenchymal stem cells (hddpscs) are loaded, and at the same time, the functional tube structure can be formed by fast flowing at body temperature, so that the medical gelatin with temperature-sensitive property and high viscosity is selected. In the second mixed solution, the selected medical gelatin concentration was 15% by mass, the photoinitiator (LAP used in this example) concentration was 0.5% by mass, and the vascular endothelial growth factor concentration was 200 ng/mL.

As shown in fig. 3, which is a rheological property diagram of the sterile pipeline phase electrospray ink and the sterile microsphere phase electrospray ink prepared by the above method in this embodiment, it can be seen from the diagram that the viscosity of 15% gelatin is much greater than that of 5% GelMA, which meets the printing requirement.

3) Extracting dental pulp mesenchymal stem cells (hDPSC) of a patient and performing functional induction:

after the wisdom tooth or the non-functional tooth of a patient is pulled out, the dental pulp of the patient is extracted in a sterile environment, and the target autologous dental pulp mesenchymal stem cell is finally obtained after operations such as cutting, digestion, identification, culture, amplification and the like. Culturing the target autologous dental pulp mesenchymal stem cells in an angiogenesis inducing culture medium, inducing for 3-7 days to differentiate hDPSC towards an angiogenesis direction, realizing the functionalization of hDPSC, and digesting to obtain the angiogenesis inducing hDPSC.

In this example, the angiogenic induction medium is EGM-2 medium comprising 5ng/mL VEGF, 5ng/mL EGF, 5ng/mL FGF, 15ng/mL IGF-1, 10mM glutamine, 0.75Unit/mL heparin, 1. mu.g/mL hydrocortisone, 50. mu.g/mL ascorbic acid and 2% fetal bovine serum.

4) Obtaining cell-carrying pipeline phase electric spraying ink:

and (3) carrying out electro-rheological jet ink on the cell-carrying pipeline by using the dental pulp mesenchymal stem cells induced by vascularization obtained in the step 3) of re-suspending the sterile pipeline electro-rheological jet water.

In this example, the density of dental pulp mesenchymal stem cells in sterile channel electrokinetic inkjet water was adjusted to 10 ten thousand/ml.

5) Assembling the coaxial double nozzles:

and inserting a 27G stainless steel nozzle into a 16G stainless steel nozzle to assemble a stainless steel coaxial double-nozzle, and treating with a nano waterproof spray to ensure the surface hydrophobicity of the nozzle, wherein the 27G nozzle is connected with an injector carrying sterile pipeline phase electric spraying ink, and the 16G nozzle is connected with an injector carrying cell pipeline phase electric spraying ink.

The coaxial inner and outer layer nozzles can be selected according to the size of the needed microspheres, in the embodiment, 27G stainless steel inner nozzles (with the outer diameter of 0.4mm and the inner diameter of 0.2mm) and 16G stainless steel outer nozzles (with the outer diameter of 1.6mm and the inner diameter of 1.16mm) are selected, so that the heterogeneous microspheres containing the pipeline structure can be manufactured integrally, and the optimal activity of the human dental pulp mesenchymal stem cells is preserved.

5) Preparation of first heterogeneous microspheres:

setting the ambient temperature to be 37 ℃, inputting an automatic preparation starting command through an upper machine position, receiving micro liquid drops formed under a high-voltage electric field by an electric spraying device, simultaneously, opening blue light of a pre-curing position to pre-cure the micro liquid drops, continuously supplying electric spraying ink by an injection pump module comprising coaxial double nozzles, starting the electric spraying ink stirring device, and finally, opening a main curing lamp of a receiving pool and finally curing the micro liquid drops to finish the preparation of the first heterogeneous microspheres.

In the electric spray preparation process, the flow rate and the used voltage of the inner spray head and the outer spray head can be adjusted according to the expected pipeline diameter, the microsphere diameter and the like, the flow rate of the inner spray head can be 2-20 mu L/min generally, the flow rate of the outer spray head can be 50-150 mu L/min, the voltage can be 2.0-4.6 kV, the microsphere diameter is continuously reduced along with the increase of the voltage, and when the voltage is applied to 4.6kV, the inner micro pipeline occupies larger space; when the flow of the outer phase spray head is constant, the higher flow of the inner phase spray head can form a micro-pipeline structure with larger diameter; when the flow of the inner phase spray head is constant, the larger the flow of the outer phase spray head is, the smaller the formed inner pipeline is. In this embodiment, the voltage is 4.6kV, the flow rate of the inner nozzle is 20 μ L/min, and the flow rate of the outer nozzle is 50 μ L/min, so as to obtain an inner phase pipe structure with a larger diameter, so that more dental pulp mesenchymal stem cells can be adhered and have functions.

6) Curing internal phase electrospray ink loaded with human dental pulp mesenchymal stem cells:

and after all the first heterogeneous microspheres are collected, uniformly transferring the dimethyl silicone oil and the first heterogeneous microspheres in the main receiving pool to a sterile centrifugal tube and putting the sterile centrifugal tube into a 4-degree refrigerator for refrigeration for 20min so as to solidify the pipe phase electric injection ink loaded with the hDPSC.

7) First heterogeneous microsphere post-treatment:

and (3) adding a proper amount of Phosphate Buffer Saline (PBS) into the completely solidified first heterogeneous microsphere-dimethyl silicone oil suspension system treated in the step 6), centrifuging for 5min at the rotating speed of 1000rpm by using a centrifuge, sucking the upper-layer dimethyl silicone oil by using a pipette after centrifuging, and repeating the operation for 3 times to complete the separation and washing of the first heterogeneous microsphere and the dimethyl silicone oil. And finally, extending the sterile injector with the metal needle into the suspension system, and guiding away residual charges in the first heterogeneous microspheres to obtain second heterogeneous microspheres.

8) Formation and functionalization of heterogeneous microsphere conduits

Placing the washed second heterogeneous microspheres into an angiogenesis induction culture medium, and placing the culture medium into a cell culture box (37 ℃, 5% CO) ₂ ) Culturing for 3-7 days to dissolve gelatin of the pipeline phase electric spraying ink, so that a micro-pipeline is formed, and the hDPSC is favorably adhered and proliferated in the pipeline to form the functionalized heterogeneous microspheres, as shown in figure 2. As can be seen from the schematic diagram, the pipes in the heterogeneous microspheres are full of hDPSC, which provides favorable conditions for subsequent implantation in the bone defect region to perform the vascularization function.

9) Application of heterogeneous microspheres

As shown in fig. 4, the obtained functionalized heterogeneous microspheres are finally assembled into a sterile injector to be used as a functionalized heterogeneous microsphere conveyor and injected into a target operation area, such as a bone defect area after tooth extraction, so that rapid vascularization and early bone formation during large-area bone reconstruction are realized. In addition, the obtained functionalized heterogeneous microspheres can also be prepared into a bone reconstruction medicament capable of realizing rapid vascularization and early bone formation.

In this embodiment, can select for use the syringe needle-free syringe of different specifications according to the size of microballon, ensure that syringe head end internal diameter is greater than heterogeneous microballon diameter, the injection process is not to the pressure of functional microballon production. In addition, according to the size and the position of the operation area, injectors with different head end lengths can be selected.

The invention aims to integrally print functionalized human dental pulp mesenchymal stem cells into a pipeline structure of heterogeneous microspheres by using a coaxial electrospray manufacturing process to obtain vascularization-promoted osteogenic microspheres, thereby facilitating the realization of rapid vascularization during large-area bone reconstruction and early formation of new bones, avoiding the complex process of cell inoculation during the conventional tissue engineering scaffold obtaining, and being a new strategy for obtaining tissue engineering microspheres.

The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.

Claims

1. A preparation method of an injectable functionalized heterogeneous microsphere is characterized by comprising the following steps:

2. The method for preparing an injectable functionalized heterogeneous microsphere according to claim 1, wherein the methacrylic acylated gelatin solution is obtained by dissolving methacrylic acylated gelatin with physiological saline under aseptic conditions; the medical gelatin solution is obtained by dissolving medical gelatin in physiological saline under aseptic conditions.

3. The method for preparing injectable functionalized heterogeneous microspheres according to claim 1, wherein the substitution rate of the methacrylic acylated gelatin in the first mixed solution is 30%, the mass concentration of the methacrylic acylated gelatin is 5%, the mass concentration of the photoinitiator is 0.5%, and the concentration of the bone morphogenic protein is 600-800 ng/mL.

4. The method for preparing injectable functionalized heterogeneous microspheres according to claim 1, wherein the photoinitiator is a blue light initiator, preferably LAP or L2959.

5. The preparation method of the injectable functionalized heterogeneous microsphere according to claim 1, wherein in the second mixed solution, the mass concentration of medical gelatin is 15%, the mass concentration of the photoinitiator is 0.5%, and the concentration of the vascular endothelial growth factor is 200-400 ng/mL.

6. The method for preparing injectable functionalized heterogeneous microspheres according to claim 1, wherein the density of the dental pulp mesenchymal stem cells in the cell-carrying conduit phase electric inkjet water is 10-15 ten thousand/ml.

7. The method of claim 1, wherein the coaxial dual nozzle is assembled by inserting a 27G stainless steel nozzle into a 16G stainless steel nozzle.

8. The method of claim 1, wherein the first nozzle has a flow rate of 50 μ L/min to 150 μ L/min, the second nozzle has a flow rate of 2 μ L/min to 20 μ L/min, and the high voltage field has a voltage of 2.0kV to 4.6 kV.

9. An injectable functionalized heterogeneous microsphere obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the injectable functionalized heterogeneous microsphere of claim 9 for the preparation of a bone-remodeling drug capable of promoting vascularization and early bone formation.