CN112791239B

CN112791239B - Preparation method of super-bionic soft and hard tissue composite scaffold

Info

Publication number: CN112791239B
Application number: CN202110049442.5A
Authority: CN
Inventors: 谢志坚; 史洋; 张淳; 石珏; 陈卓; 贺永; 孙元; 喻康; 吕尚
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-01-14
Filing date: 2021-01-14
Publication date: 2021-10-26
Anticipated expiration: 2041-01-14
Also published as: CN112791239A

Abstract

The invention discloses a preparation method of a super-bionic soft and hard tissue composite scaffold, which comprises the steps of designing a bionic bone tissue scaffold, preparing printing raw materials, preparing printing ink, DLP (digital light processing) photocuring 3D printing, freeze-drying, extracting dental pulp mesenchymal stem cells of a patient, directionally differentiating, inoculating and culturing the cells, preparing a soft tissue scaffold and preparing the soft and hard tissue composite scaffold. According to the invention, the 3D printing of the super-bionic biomaterial is adopted to realize the synchronous personalized repair of soft and hard tissues in the oral clinic, so that the development of a second operation area is avoided, and the treatment time for the staged repair of the soft and hard tissues at present is shortened. Meanwhile, inspired by the three-dimensional microstructure of the jaw bone tissue, the Haffman tube and the Wolman tube of the jaw bone are simulated to establish good blood supply at an early stage, conditions are provided for layering induction of the bionic jaw bone tissue, and a new thought is provided for design and research and development of the bionic scaffold.

Description

Preparation method of super-bionic soft and hard tissue composite scaffold

Technical Field

The invention belongs to a preparation method of a bionic scaffold in the field of tissue engineering, and particularly relates to a preparation method of a super-bionic soft and hard tissue composite scaffold.

Background

In general, 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. Clinically, however, defects in the oral bone tissue, especially the insufficient amount of alveolar bone over a wide range, will directly affect the patient's prosthetics treatment, i.e., the reconstruction of masticatory 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. Furthermore, the jaw bone defect of the oral cavity is mostly accompanied with the recession of soft tissues, when the soft tissues of the gum, especially the attached gum, are insufficient, because the soft tissues lack submucosa, the soft tissue tension generated in the bone augmentation operation is greatly increased, and great challenges are provided for the operation process and the long-term curative effect. The most common clinical means for treating soft and hard tissue combined defects at present is to reconstruct jaw bone through a first-stage bone augmentation operation, such as bone grafting, bone splitting and the like, and then achieve the purpose of soft and hard tissue regeneration and repair through a second-stage autologous soft tissue transplantation operation, so as to provide an anatomical basis for subsequent treatment and prognosis, but the treatment process also has the problems of more operation times, long period, need to open a second operation area and the like. Therefore, the problem of repairing the defects of the oral jaw and face is the synchronous reconstruction treatment of large-area bone tissue defects and soft tissue deficiency, and the development of soft and hard tissue composite tissue engineering materials for synchronous repair is the important clinical requirement of the oral cavity at present.

In the field of bone tissue engineering, biological scaffold materials that are widely used are mainly focused on bioceramics, natural or synthetic polymers and composites. However, most of the existing scaffolds for bone tissue engineering have the disadvantage of simple and uniform structure, and from the histological point of view, the natural jaw bone is composed of high-density cortical bone on the surface and cancellous bone inside, the former uses densely arranged bone units as basic elements and is the main source of mechanical strength of bone tissue; the latter is directly constituted by the cancellous network trabeculae. Furthermore, in cortical bone, the haversian canals and the Wolkman canals rich in blood vessels and nerves are criss-crossed to form nutrition network traffic, and play an indispensable role in bone reconstruction and repair; the cancellous bone has loose and porous structure and abundant blood circulation, and the structural characteristics ensure the physiological function and adaptability reconstruction of the natural jaw bone. When the bone tissue is reconstructed, the reconstructed size of more than 200 μm exceeds the diffusion limit of nutrients and oxygen, and a nutrient network needs to be constructed to avoid unsmooth internal nutrient transportation. Therefore, the key element in the ideal repair of standard bone defects of complex morphology is to fully mimic the microstructure of natural bone tissue and to form an effective vascular network within the bone scaffolding material at an early stage to ensure oxygen and nutrient exchange in the central portion. And the high-precision 3D printing technology provides possibility for simulating the bone tissue of the natural nano-micro structure in bone tissue engineering. In addition, because the jaw bone needs to realize the force transmission through the occlusion of teeth and needs to have good adaptive reconstruction function under the action of the occlusion force, the jaw bone has more unique requirements on the structure and the connection mode of trabecula bone in cancellous bone, but the functional characteristics of the biomaterial for repairing the jaw bone are rarely emphasized at present and only the morphological reconstruction is concerned, so that the design and the manufacture of the bone tissue engineering scaffold material which is specific to the jaw bone reconstruction in the oral cavity field to adapt to the oral environment and the functions are the bottleneck of the current research. If the graft material can fully simulate the vascular structure of natural jaw bone tissue, the blood supply is ensured, meanwhile, the multidirectional differentiation potential stem cells from the patient are loaded, the new bone tissue can be induced to play the oral physiological function which the new bone tissue needs, the immunogenicity risk caused by heterologous cells does not exist, and no scholars can simulate the microstructure of the natural jaw bone to prepare the jaw bone scaffold material with larger size from the view of microcosmic and clinical transformation at present.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a preparation method of a super-bionic soft and hard tissue composite scaffold. The invention provides a new strategy for biomimetically repairing the jaw bone by gradient mineralization of a multi-layer bionic through analyzing a vascular network of a haversian tube and a Wolkman tube in a natural jaw cortical bone and a three-dimensional porous composite microstructure of a cancellous bone which is complex and changeable in adaptation to oral occlusal force, and constructs a super-bionic soft and hard tissue composite scaffold by combining a high-precision electro-hydrodynamics printing technology and a high-precision DLP photocuring 3D printing technology to realize synchronous repair of oral soft and hard tissues.

The invention adopts the following specific technical scheme:

a preparation method of a super-bionic soft and hard tissue composite scaffold comprises the following steps:

1) simulating a jaw bone tissue microstructure through three-dimensional software to obtain a bionic thin-layer cortical bone scaffold model and a cancellous bone scaffold model;

2) dissolving GelMA by using normal saline, then uniformly dissolving a photoinitiator and BMP-2 in GelMA solution in sequence, and adjusting the pH of the solution to be neutral to obtain cortical bone GelMA pre-polymerization solution and cancellous bone GelMA pre-polymerization solution respectively;

3) respectively printing the cortical bone GelMA pre-polymerized liquid and the cancellous bone GelMA pre-polymerized liquid obtained in the step 2) on the cortical bone support model and the cancellous bone support model in the step 1) through photocuring and layering, and cleaning the GelMA which is not crosslinked and cured in the printing process to respectively obtain a cortical bone preparation support with a hollow vascular network flow channel and a spongy cancellous bone preparation support with a plurality of porous channels;

4) placing the cortical bone prepared support and the cancellous bone prepared support obtained in the step 3) in an ultralow temperature environment to enable water molecules in the prepared support to rapidly form ice crystals; then placing the mixture in a low-temperature environment for tempering so that ice crystals grow in a fusion manner and form a communicated microstructure; finally, gradient heating, vacuum drying to remove moisture, and sterilizing to obtain a cortical bone scaffold and a cancellous bone scaffold;

5) respectively culturing the target autologous dental pulp mesenchymal stem cells in an osteogenic induction culture medium and an angiogenisis induction culture medium, inducing for 7-14 days to make the target autologous dental pulp mesenchymal stem cells differentiate into osteogenic cells and angiogenisis, and digesting and resuspending to obtain osteogenic induction hDPSC and angiogenisis induction hDPSC;

6) inoculating the vascularization induced hDPSC obtained in the step 5) into a hollow vascular network flow channel of the cortical bone scaffold, inoculating the vascularization induced hDPSC on the upper surface and the lower surface of the cortical bone scaffold, and culturing to enable cells to be spread over the whole cortical bone scaffold, thereby obtaining a functional cortical bone scaffold; mixing osteogenic induced hDPSC and vascularization induced hDPSC, inoculating the mixture on a cancellous bone scaffold, and culturing to ensure that cells are spread over the whole cancellous bone scaffold to obtain a functionalized cancellous bone scaffold;

7) printing polycaprolactone to form a net-shaped support structure by an electrofluid mechanics printing technology; dissolving GelMA with normal saline, and then adding a photoinitiator to obtain soft tissue layer pre-polymerization liquid; placing the mesh-shaped scaffold structure in a prepolymerization solution, forming the mesh-shaped scaffold structure through photocuring, and sterilizing to obtain a soft tissue scaffold;

8) dissolving GelMA in normal saline under aseptic condition, and adding a photoinitiator to obtain a prepolymerization forming solution; and (3) sequentially overlapping the soft tissue scaffold, the functional cortical bone scaffold and the functional cancellous bone scaffold from top to bottom, uniformly dropwise adding the pre-polymerization forming liquid at an overlapping interface, and carrying out photocuring to integrally form the three so as to obtain the super-bionic soft and hard tissue composite scaffold.

Preferably, the GelMA substitution rate in the cortical bone GelMA pre-polymerization solution is 90%, the mass concentration is 10%, the mass concentration of the photoinitiator is 0.5%, and the concentration of BMP-2 is 600-800 ng/mL; the GelMA pre-polymerization liquid for cancellous bone has the GelMA substitution rate of 60%, the mass concentration of 10%, the mass concentration of a photoinitiator of 0.5%, and the concentration of BMP-2 of 200-400 ng/L.

Further, the photoinitiator is a blue light initiator LAP.

Preferably, the three-dimensional software in the step 1) is SolidWorks software; adjusting the pH value of the solution to 7.4 in the step 2); the sterilization was irradiated with Co60 for 24 hours.

Preferably, in the step 3), the photocuring layered printing of the cortical bone GelMA prepolymer solution and the cancellous bone GelMA prepolymer solution is realized by a DLP photocuring 3D printer, and the uncrosslinked and solidified GelMA in the printed cortical bone preparation scaffold and cancellous bone preparation scaffold is washed by a physiological saline solution containing isotonic BMP-2 in a shaking table, and the remaining photoinitiator which is not crosslinked is leached.

Preferably, in the step 4), the cortical bone preparation support and the cancellous bone preparation support are placed in a refrigerator at-80 ℃ for rapid cooling and are kept for 3 hours, so that water molecules in the preparation support rapidly form ice crystals; then placing the mixture in a refrigerator at the temperature of minus 20 ℃ for tempering for 5 hours to ensure that ice crystals are fused and grown and form a communicated microstructure; and finally, placing the scaffold in a freeze dryer for gradient temperature rise, carrying out vacuum drying for 24 hours to remove moisture, and sterilizing to obtain the cortical bone scaffold and the cancellous bone scaffold.

Preferably, the osteogenesis induction medium is an alpha-MEM medium, and comprises 100nmol/L dexamethasone, 5mmol/L beta-sodium glycerophosphate, 100 mu g/mL ascorbic acid, 10% fetal bovine serum by mass, 100Units/mL penicillin and 100Units/mL streptomycin; the angiogenesis induction medium is EGM-2 medium, and comprises 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.

Preferably, in the step 6), the culture time is 7-14 days; mixing osteogenic induction hDPSC and vascularization induction hDPSC according to the density ratio of 1:1, and then inoculating the mixture on a spongy bone scaffold.

Preferably, in the step 7), firstly, polycaprolactone particles are filled into a charging barrel of the direct writing device, then the polycaprolactone particles are preheated for 1-3 hours at 80-100 ℃ so that the polycaprolactone particles are fully melted and filled into the charging barrel, and then printing is performed; the parameters of the printing process are as follows: the distance between the needle head and the substrate is 2mm, the diameter of the needle head is 150 mu m, the temperature of the needle head is 105 ℃, the temperature of the material barrel is 105 ℃, and the moving speed is 700-; the substitution rate of GelMA in the soft tissue layer pre-polymerization liquid is 60%, the mass concentration is 10%, and the mass concentration of the photoinitiator is 0.5%; the time for photocuring is 15 seconds or more.

Preferably, in the step 8), the substitution rate of GelMA in the prepolymerization molding solution is 90%, and the mass concentration is 10%; the photocuring time was 5 seconds.

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

1) the invention can construct and obtain the soft and hard tissue composite scaffold by utilizing the photocuring crosslinking performance of GelMA, and can repair soft and hard tissues of the oral cavity simultaneously while solving the problem of difficult soft tissue closure after the bone augmentation surgery.

2) The invention induces the new bone to naturally form the bone density which is in phase transition and consistent with the adjacent old bone by regulating the concentration difference of BMP-2 in the cortical bone layer and the cancellous bone layer in the bone tissue bracket, thereby realizing the functional density gradient reconstruction of the new bone tissue.

3) The invention utilizes high-precision electro-hydrodynamics printing technology to prepare the PCL network, and the grid density can be adjusted through different equipment parameters to realize the suturable performance of the soft tissue bracket, which cannot be achieved by the soft tissue bracket on the market at present.

4) The dental pulp mesenchymal stem cells of a patient are utilized to carry out directional induction and differentiation and are inoculated to the scaffold in vitro to obtain the tissue engineering scaffold, so that on one hand, the tissue engineering scaffold can quickly establish blood supply in vivo and induce the early formation of new bones, on the other hand, the problem of immunogenicity is avoided while the autologous tissues are changed into valuable things, and the clinical transformation is facilitated.

Drawings

FIG. 1 shows a cortical bone scaffold model (A), a cancellous bone scaffold model (B) and a composite bone tissue scaffold model (C) after the cortical bone scaffold model and the cancellous bone scaffold model are combined, which are designed by SolidWorks software according to the present invention;

FIG. 2 is a schematic view of a PCL mesh stent structure;

FIG. 3 is a schematic structural diagram of the super-bionic soft and hard tissue composite scaffold of the invention;

FIG. 4 is a graph of the results of a stress-strain experiment for a soft tissue scaffold.

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.

The invention provides a preparation method of a super-bionic soft and hard tissue composite scaffold, which comprises the following specific steps:

1) designing a bionic bone tissue scaffold:

jaw bone tissue microstructures are simulated through three-dimensional software (such as SolidWorks software), and a bionic thin-layer cortical bone scaffold model and a cancellous bone scaffold model are designed and obtained. As shown in fig. 1, an embodiment of the present invention provides a cortical bone scaffold model (a), a cancellous bone scaffold model (B), and a composite bone tissue scaffold model (C) after combining the two. Wherein, the cortical bone scaffold model contains rich interactive vascular networks, is convenient for inoculating the differentiated human tooth marrow mesenchymal stem cells (namely hDPSC) from blood vessels in the later period, establishes blood supply in the early period and promotes the formation of new bones. The spongy bone scaffold model is a loose and porous spongy structure, and the mutually staggered and communicated porous structures are favorable for early adhesion and creeping of human dental pulp mesenchymal stem cells differentiated from osteogenesis and angiogenisis.

2) Preparation of printing ink:

GelMA (i.e., methacrylated gelatin) was first dissolved with physiological saline, and then the photoinitiator and BMP-2 were uniformly dissolved in the GelMA solution in this order. Because the solution is weakly acidic, the pH value of the solution is adjusted to be neutral by adding a proper amount of sodium hydroxide, and a cortical bone GelMA pre-polymerization solution and a cancellous bone GelMA pre-polymerization solution are respectively obtained.

In the embodiment, the GelMA substitution rate in the cortical bone GelMA pre-polymerization solution is 90%, the mass concentration is 10%, the mass concentration of the photoinitiator is 0.5%, and the concentration of BMP-2 is 600-800 ng/mL (preferably 800 ng/mL). The GelMA pre-polymerization liquid for cancellous bone has the GelMA substitution rate of 60 percent, the mass concentration of 10 percent, the mass concentration of 0.5 percent of photoinitiator and the concentration of BMP-2 of 200-400 ng/mL. The photoinitiator can be blue light initiator LAP, and the pH of the solution is adjusted to 7.4.

Because the density of the induced new bone has concentration dependency when the BMP-2 is in a safe use range, namely the density of the formed new bone is higher when the BMP-2 is in the safe use range, the density of the new bone is higher, the new bone is induced to naturally form bone density which is in phase shift and consistent with that of the adjacent old bone by regulating and controlling the concentration difference of the BMP-2 in a cortical bone layer and a cancellous bone layer in a bone tissue scaffold, and the functionalized density gradient reconstruction of the new bone tissue is realized.

In addition, the adjustable range of the physical and chemical properties of GelMA is large, GelMA with different substitution rates and different concentrations can be selected according to needs so as to meet different needs, therefore, the substitution rate and the concentration of the selected GelMA in the design are relatively optimal, the requirements of simultaneous printability, biocompatibility and degradability can be met, and the GelMA can be adjusted under the condition of needs. For example, in a soft tissue scaffold layer, the mechanical properties can be further increased by selecting a GelMA substitution rate of 90% and a concentration of 15%.

3) DLP photocuring 3D printing:

loading the cortical bone GelMA pre-polymerization liquid obtained in the step 2) into an ink tank of a DLP photocuring 3D printer, printing a cortical bone scaffold model designed on SolidWorks software in the step 1) by photocuring GelMA layering, taking out the scaffold after printing, placing the scaffold into a normal saline solution containing isotonic BMP-2, washing the GelMA which is not crosslinked and cured in the printing process of the scaffold by a shaking table, smoothing a hollow vascular network flow channel structure in the scaffold, and leaching out an uncrosslinked photoinitiator.

Similarly, the spongy bone GelMA pre-polymerization liquid obtained in the step 2) is filled into an ink tank of a DLP photocuring 3D printer, the spongy bone support model designed in the step 1) on SolidWorks software is printed by photocuring GelMA in a layering mode, the support is taken out after printing is finished, the support is placed in a normal saline solution containing isotonic BMP-2, GelMA which is not crosslinked and cured in the printing process of the support is cleaned by a shaking table, so that a porous structure in the support is loosened, and meanwhile, an uncrosslinked photoinitiator is leached.

And finally obtaining a cortical bone prepared scaffold with a hollow vascular network flow channel and a spongy porous spongy cancellous bone prepared scaffold by DLP photocuring 3D printing.

4) And (3) freeze drying:

placing the cortical bone prepared support and the cancellous bone prepared support obtained in the step 3) in an ultralow temperature environment to enable water molecules in the prepared support to rapidly form ice crystals. And then tempering in a low-temperature environment to ensure that the ice crystals grow in a fusion manner, wherein the molecular network microstructure exists in the photocured part of the preliminary support, so that the molecular network microstructure can be punctured in the process of ice crystal fusion growth to form a communicated channel microstructure. And finally, gradient heating and vacuum drying to remove moisture, thereby obtaining the cortical bone scaffold and the cancellous bone scaffold.

In this example, the cortical bone and cancellous bone scaffolds were placed in a refrigerator at-80 ℃ for rapid cooling and maintained at that temperature for 3 hours, so that water molecules inside the scaffolds rapidly formed dense and fine ice crystal particles. And then the bracket is placed in a refrigerator at the temperature of 20 ℃ below zero to be tempered for 5 hours, so that the ice crystal particles are fused and grown, and a communicated channel microstructure is formed. And finally, placing the scaffold in a freeze dryer for gradient temperature rise, and performing vacuum drying for 24 hours to remove moisture to obtain the cortical bone scaffold and the cancellous bone scaffold. And (3) placing the cortical bone scaffold and the cancellous bone scaffold under Co60 irradiation condition for 24 hours for sterilization, and storing for later use.

5) Extracting dental pulp mesenchymal stem cells of a patient and directionally differentiating:

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.

Respectively culturing the target autologous dental pulp mesenchymal stem cells in an osteogenic induction culture medium and an angiogenisis induction culture medium, inducing for 7-14 days to ensure that the target autologous dental pulp mesenchymal stem cells are differentiated into osteogenesis and angiogenisis, realizing the functionalization of the target autologous dental pulp mesenchymal stem cells, and obtaining osteogenic induction hDPSC and angiogenization induction hDPSC after digestion and heavy suspension.

In this example, the osteogenesis induction medium was α -MEM medium containing 100nmol/L dexamethasone, 5mmol/L sodium β -glycerophosphate, 100. mu.g/mL ascorbic acid, 10% fetal bovine serum by mass, 100Units/mL penicillin and 100Units/mL streptomycin. The angiogenesis inducing medium is EGM-2 medium, which comprises 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.

6) Inoculation and culture of cells:

inoculating the vascularization induced hDPSC obtained in the step 5) into a hollow vascular network flow channel of the cortical bone scaffold, inoculating the vascularization induced hDPSC on the upper surface and the lower surface of the cortical bone scaffold, and culturing to enable cells to be spread over the whole cortical bone scaffold, thereby obtaining the functional cortical bone scaffold. The process is as follows: firstly, a cortical bone scaffold is horizontally placed on an operation table, vascularization induced hDPSC is inoculated in a hollow vascular network flow channel of the cortical bone scaffold, vascularization induced hDPSC is inoculated on the upper surface of the cortical bone scaffold, and the vascularization induced hDPSC is adhered to the inner lower surface of the hollow vascular network flow channel under the action of gravity and proliferated and grown on the surface, but the vascularization induced hDPSC is difficult to spread over the whole hollow vascular network flow channel. Therefore, after one day of culture, the cortical bone scaffold is turned over by 180 degrees, the original lower surface of the cortical bone scaffold is placed upwards, the same inoculation operation is carried out, namely, the vascularization induced hDPSC is inoculated on the original upper surface of the hollow vascular network flow channel of the cortical bone scaffold, and the vascularization induced hDPSC is inoculated on the upper surface of the cortical bone scaffold, so that cells can be spread over the whole scaffold, and the cortical bone scaffold is cultured in a culture medium for 7-14 days for later use.

And (3) mixing the osteogenesis induced hDPSC and the vascularization induced hDPSC, inoculating the mixture on the cancellous bone scaffold, and culturing to ensure that the cells are distributed throughout the cancellous bone scaffold to obtain the functionalized cancellous bone scaffold. In this embodiment, the osteogenesis-induced hddpscs and the vascularization-induced hddpscs may be mixed at a density ratio of 1:1, and then seeded on a cancellous bone scaffold, and cultured in a culture medium for 7-14 days to allow the cells to proliferate and spread throughout the scaffold for use. In this example, mixing osteogenic and vascularization-induced hddpscs in a density ratio of 1:1 resulted in better biological performance, and enhanced osteogenic and vascularization functions without affecting their growth.

The functionalized cortical bone scaffold inoculated with the cells for 7-14 days is characterized by a laser scanning confocal microscope, and a confocol diagram shows that the cells are densely adhered in a vascular flow channel to outline a vascular-like structure, and a large number of cells are also visible on the upper surface and the lower surface of the scaffold and in the solid part of the scaffold, which indicates that the cells can be uniformly adhered and proliferated on the inner surface and the outer surface of the cortical bone scaffold.

The functionalized cancellous bone scaffold inoculated with the cells for 7-14 days is characterized by a laser scanning confocal microscope, and a confocol diagram shows that a large number of cells are densely adhered to the inner and outer surfaces of the pores of the cancellous bone scaffold, so that the porous shape of the cancellous bone scaffold is outlined, and the cells can be uniformly adhered to and proliferate on the inner and outer surfaces of the cancellous bone scaffold.

7) Preparing a soft tissue scaffold:

and a net-shaped support structure is formed by printing polycaprolactone through a high-precision electro-hydrodynamics printing technology. GelMA is dissolved by normal saline, and then a photoinitiator is added to obtain the soft tissue pre-polymerization solution. And (3) placing the reticular stent structure in the soft tissue pre-polymerization liquid, and forming the reticular stent structure through photocuring to obtain the soft tissue stent.

In this embodiment, through high accuracy electro-hydrodynamics printing technique, pack into Polycaprolactone (PCL) granule in the metal feed cylinder of direct writing equipment, preheat polycaprolactone granule 1 ~ 3 hours under the condition of 80 ~ 100 ℃ afterwards, make polycaprolactone granule fully melt and fill the feed cylinder. Then, formal printing is started, and parameters in the printing process are as follows: the distance between the needle head and the substrate is 2mm, the diameter of the needle head is 150 μm, the temperature of the needle head is 105 ℃, the temperature of the material cylinder is 105 ℃, the moving speed is 700-. Then, a molding solution was prepared in which the substitution rate of GelMA was 60%, the mass concentration was 10%, and the mass concentration of the photoinitiator was 0.5%. And pouring the molding solution into a molding container, finally placing the reticular stent structure in the molding container, and performing photocuring for more than 15 seconds to shape the reticular stent structure to obtain the soft tissue stent. And (3) placing the soft tissue stent under Co60 irradiation for 24 hours for sterilization, and storing for later use.

8) Preparing a soft and hard tissue composite scaffold:

GelMA and a photoinitiator are dissolved by normal saline under aseptic conditions to obtain a prepolymerization forming solution. The substitution rate of GelMA in the prepolymerization forming solution is 90 percent, and the mass concentration is 10 percent. The photocuring time was 5 seconds. Sequentially overlapping the soft tissue scaffold, the functional cortical bone scaffold and the functional cancellous bone scaffold from top to bottom, completely and uniformly dripping a prepolymerization forming liquid at an overlapping interface, and carrying out photocuring to integrally form the three to obtain the super-bionic soft and hard tissue composite scaffold, which is shown in figure 3. The soft tissue scaffolds which can be sutured are sequentially arranged from top to bottom, the middle layer is a cortical bone scaffold which is continuous with adjacent cortical bone and contains a functional vascular network, and the lower layer is a functional cancellous bone scaffold which is continuous with adjacent cancellous bone.

The obtained soft tissue scaffold was subjected to a stress-strain experiment to verify its mechanical properties, and the results are shown in fig. 4. GM60 is a non-reinforced soft tissue scaffold prepared directly from a soft tissue pre-polymerization solution without a mesh scaffold structure; composite is a PCL-reinforced soft tissue scaffold prepared by the method of the invention. As can be seen from the figure, the composite containing the PCL reticular scaffold structure has better mechanical property compared with GM60, thereby endowing the soft tissue scaffold with suturable performance, and the soft tissue is directly sutured with the gingival tissue around the soft and hard tissue defect area of the oral cavity, so that the soft and hard tissue can be repaired simultaneously, and the second operation area can be avoided.

The invention discloses a preparation method of a super-bionic soft and hard tissue composite scaffold combining a high-precision electro-hydrodynamics printing technology and a high-precision DLP photocuring 3D printing technology. The soft tissue scaffold is mainly composed of methacrylic acid hydrogel (GelMA), the interior of the soft tissue scaffold contains a Polycaprolactone (PCL) enhanced network formed by high-precision electro-hydrodynamics printing, and the soft tissue scaffold has good biocompatibility and suturability; the double-layer bone tissue scaffold is composed of a cortical bone scaffold with an upper layer containing a vascular network and a spongy porous cancellous bone scaffold with a lower layer, and the gradient density of new bones is controlled by bone morphogenetic protein-2 (BMP-2) with gradient concentration in the double-layer scaffold so as to induce the super-bionic reconstruction of jaw bone tissues.

The invention aims to realize the synchronous personalized repair of soft and hard tissues in the oral clinic by 3D printing of the super-bionic biomaterial, thereby not only avoiding the development of a second operation area, but also shortening the treatment time of the current soft and hard tissue staged repair. Meanwhile, inspired by the three-dimensional microstructure of the jaw bone tissue, the Haffman tube and the Wolman tube of the jaw bone are simulated to establish good blood supply at an early stage, conditions are provided for layering induction of the bionic jaw bone tissue, and a new thought is provided for design and research and development of the bionic scaffold.

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 a super-bionic soft and hard tissue composite scaffold is characterized by comprising the following steps:

2. The preparation method according to claim 1, wherein the GelMA pre-polymerization solution of the cortical bone has a GelMA substitution rate of 90%, a mass concentration of 10%, a mass concentration of 0.5% of a photoinitiator, and a BMP-2 concentration of 600-800 ng/mL; the GelMA pre-polymerization liquid for cancellous bone has the GelMA substitution rate of 60%, the mass concentration of 10%, the mass concentration of a photoinitiator of 0.5%, and the concentration of BMP-2 of 200-400 ng/L.

3. The method of any one of claims 1 or 2, wherein the photoinitiator is a blue light initiator LAP.

4. The preparation method according to claim 1, wherein the three-dimensional software in the step 1) is SolidWorks software; adjusting the pH value of the solution to 7.4 in the step 2); the sterilization was irradiated with Co60 for 24 hours.

5. The preparation method according to claim 1, wherein the step 3) is implemented by a DLP photocuring 3D printer to perform photocuring layered printing of the cortical bone GelMA pre-polymerization solution and the cancellous bone GelMA pre-polymerization solution, and the uncrosslinked and cured GelMA in the printed cortical bone preparation scaffold and cancellous bone preparation scaffold is washed by a physiological saline solution containing isotonic BMP-2 in a shaking table, and the remaining photoinitiator which is not crosslinked is leached out.

6. The preparation method according to claim 1, wherein in the step 4), the cortical bone preparation support and the cancellous bone preparation support are placed in a refrigerator at-80 ℃ for rapid cooling and are kept for 3 hours, so that water molecules in the preparation supports rapidly form ice crystals; then placing the mixture in a refrigerator at the temperature of minus 20 ℃ for tempering for 5 hours to ensure that ice crystals are fused and grown and form a communicated microstructure; and finally, placing the scaffold in a freeze dryer for gradient temperature rise, carrying out vacuum drying for 24 hours to remove moisture, and sterilizing to obtain the cortical bone scaffold and the cancellous bone scaffold.

7. The method of claim 1, wherein the osteogenesis inducing medium is an α -MEM medium comprising 100nmol/L dexamethasone, 5mmol/L sodium β -glycerophosphate, 100 μ g/mL ascorbic acid, 10% fetal bovine serum by mass, 100Units/mL penicillin and 100Units/mL streptomycin; the angiogenesis induction medium is EGM-2 medium, and comprises 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.

8. The method according to claim 1, wherein in the step 6), the culturing is carried out for 7 to 14 days; mixing osteogenic induction hDPSC and vascularization induction hDPSC according to the density ratio of 1:1, and then inoculating the mixture on a spongy bone scaffold.

9. The preparation method according to claim 1, wherein in the step 7), polycaprolactone particles are firstly filled into a cylinder of a direct writing device, and then the polycaprolactone particles are preheated for 1-3 hours at 80-100 ℃ so as to be fully melted and filled into the cylinder, and then printing is carried out; the parameters of the printing process are as follows: the distance between the needle head and the substrate is 2mm, the diameter of the needle head is 150 mu m, the temperature of the needle head is 105 ℃, the temperature of the material barrel is 105 ℃, and the moving speed is 700-; the substitution rate of GelMA in the soft tissue layer pre-polymerization liquid is 60%, the mass concentration is 10%, and the mass concentration of the photoinitiator is 0.5%; the time for photocuring is 15 seconds or more.

10. The process according to claim 1, wherein in the step 8), the substitution rate of GelMA in the preliminary polymerization molding liquid is 90% and the mass concentration is 10%; the photocuring time was 5 seconds.