CN110559483A

CN110559483A - Design and application of cancellous bone bionic scaffold prepared by 3d printing technology

Info

Publication number: CN110559483A
Application number: CN201810576365.7A
Authority: CN
Inventors: 孙晗笑; 利时雨
Original assignee: Guangzhou Abduction Biotechnology Co Ltd
Current assignee: Guangzhou Abduction Biotechnology Co Ltd
Priority date: 2018-06-06
Filing date: 2018-06-06
Publication date: 2019-12-13

Abstract

The invention constructs a novel bionic scaffold model by a 3D-Voronoi algorithm and a random distribution micropore algorithm, and manufactures the high-precision tissue engineering scaffold by applying a biological 3D printing technology. The bovine femur is calcined by using an ammonium dihydrogen phosphate secondary calcination method, inorganic components such as hydroxyapatite are removed on the basis of realizing the degreasing, decalcification and antigen removal of the allogeneic bone, the remainder is high-purity beta-tricalcium phosphate, the form of the natural cancellous bone is reserved, and the variable difference caused by the matrix material in the experiment is eliminated. Through various experiments such as characterization detection, structural analysis, in vitro experiments, in vivo implantation and the like, the influence of different scaffold structures on cell proliferation adhesion and in vivo bone repair effects is comprehensively evaluated, a reliable scaffold model and an effective detection means are provided for the bionic structure design of the bone tissue engineering scaffold, and the scaffold can be used for various clinical bone defects.

Description

Design and application of cancellous bone bionic scaffold prepared by 3d printing technology

Technical Field

The invention belongs to the field of tissue engineering, and particularly relates to application of a novel bionic scaffold model.

Background

The skeletal system of the human body has important functions of maintaining movement, protecting internal organs, supporting body weight and the like. Many bone defect cases are caused by trauma, infection, tumor, surgical excision and the like every year in China, and the bone defect repair faces huge clinical requirements and shows a continuously rising trend. The bone defect has many repairing materials, which can be classified into autologous materials, allogenic materials and artificial materials according to the source.

The autologous bone transplantation is a gold standard for treating bone defects, has the advantages of no immunological rejection, complete absorption and effective induction of bone reconstruction, and can be used for filling and repairing a small amount of bone defects clinically at present by using bone fragments and bone powder; the major mandibular defect repair is mainly used for transplanting after the vascularized iliac valve, fibula valve or non-vascularized iliac and rib are free. However, since the source of autologous bone is limited, a second operation area needs to be opened up to create new bone defect, which easily causes complications such as infection, hematoma and nerve injury, and secondary injury and new bone defect are easy to occur in the repair process, so that the clinical application is limited, and the autologous bone transplantation cannot meet huge clinical requirements. Allogeneic bone is relatively more widely available than autologous bone, and is usually processed to reduce antigenicity and facilitate storage, but even then, some patients experience rejection of the allogeneic bone, and resorption and nonunion.

The artificial material is used as a novel bone defect repairing material, can effectively realize the functions of bone defect filling and structure supporting, solves the problems of limited bone supply amount, immune rejection reaction avoidance, easy shaping and convenient matching of the anatomical structure of a defect area. Clinically, the medical artificial bone repair material can be divided into a metal material and a non-metal material. The medical metal material comprises stainless steel, titanium-based alloy, cobalt-based alloy and the like. Taking titanium alloy as an example, the titanium alloy has good mechanical strength, good biocompatibility and low postoperative infection rate. During operation, a doctor can manually cut the titanium mesh plate according to the size and the shape of the defect part of a patient, manually shape the titanium mesh plate and fix the titanium mesh plate by using screws to realize defect repair. The titanium mesh which is attached to the anatomical structure of the skull of the patient can be well adjusted before the operation by combining the three-dimensional reconstruction technology, thereby greatly shortening the operation time.

The repair and healing of bone defect is a complex pathological and physiological process, and the bone tissue engineering scaffold has certain requirements on the matrix material and the three-dimensional structure of the scaffold in order to effectively load growth factors and seed cells, actively promote bone growth, angiogenesis and nutrient metabolism after implantation, avoid infection, immunological rejection and other problems. In summary of previous research perspectives, an ideal scaffold for bone tissue engineering should have the following properties: (1) the material has good plasticity and certain mechanical strength, so that the material can maintain the morphological structure within a certain time and is matched with the biomechanical property of the original bone tissue of the implanted part; (2) good biocompatibility and surface activity, and is beneficial to the adhesion, proliferation and extracellular matrix secretion of seed cells on the surface of the material; (3) good bone conductivity, which is beneficial to the growth of new bone tissues and vascular tissues; (4) good bone inductivity, can stimulate cells around the implantation position on the material to differentiate to cartilage cells and osteoblasts to form new bone tissues; (5) has three-dimensional porous structure, communicated micropores and higher porosity, and is beneficial to the adhesion of seed cells and growth factors and the exchange of nutrient substances and metabolites.

The 3D printing technology is also called a rapid prototyping technology or an additive manufacturing technology, and is a manufacturing technology for manufacturing a three-dimensional real object by printing a bondable material layer by layer and superimposing the bondable material layer by layer on the basis of a digital three-dimensional model. In recent years, 3D printing technology has the advantages of rapid molding, precise replication, complex structure, etc., and is gradually favored by researchers in the biomedical field and applied, specifically including fabrication of implant prostheses, surgical instruments, surgical guide plates, printing of biological cells, etc. The 3D printing technology is used for constructing the bone tissue engineering scaffold, and the following advantages of the 3D printing technology can be embodied: (1) a complex three-dimensional structure can be designed as a printing template by using three-dimensional design software; (2) accurately printing isometric three-dimensional real objects based on the digital three-dimensional model, and well matching the anatomical morphology of bone tissues; (3) the internal structure and porosity of the porous scaffold can be accurately controlled; (4) the material can be printed with various materials, such as high molecular materials, such as polylactic acid (PLA), polyglycolic acid, Polycaprolactone (PCL), etc., biological ceramics, such as Hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP), Bioactive Glass (BG), etc., metal materials, such as titanium-based alloy, cobalt-based alloy, etc., biological materials, such as histiocytes.

As an emerging class of 3D printing, a biological 3D printer capable of printing living cells and bioactive materials provides the possibility of fabricating high-precision tissue engineering scaffolds. Among various biological 3D printers, the pneumatic extrusion type 3D printer is favored by enterprises and scientific research institutions due to the advantages of convenience in material storage, no damage to cell materials, high printing precision, high molding speed, no pollution in the molding process and the like, and particularly the pneumatic extrusion type biological 3D printer does not relate to chemical changes in the molding preparation process, so that the molded entity keeps the original physicochemical property. The multifunctional large printing nozzle can be configured on the pneumatic extrusion type biological 3D printer according to requirements, in a conventional manufacturing process, the printing nozzle has a temperature control function within a certain range, pneumatic pressure is applied to gelatinous and pasty raw materials at a proper temperature according to requirements, the materials are extruded out of the printing nozzle, a numerical control motor prints out a specific layer of shape according to a preset moving track, then a plane is raised to print a second layer, and the layers are stacked layer by layer in sequence, so that a three-dimensional structure is built on a working platform. The preparation of the three-dimensional porous scaffold of the artificial bone repair material is realized by utilizing a 3D printing technology, is a leading-edge and important part in bone repair engineering, can load stem cells, is beneficial to the growth and differentiation of the stem cells, promotes the formation of new bone tissues under specific conditions, improves the bone formation effect and realizes a good bone repair effect.

The seed cell is an indispensable important part for constructing the engineered tissue, and provides the physiological function of a specific cell type for the tissue engineering scaffold by the proliferation and differentiation of the seed cell into the specific cell type. Therefore, obtaining seed cells of appropriate type, sufficient number, vigorous proliferation, and not causing immune rejection is a prerequisite and basis for the construction of engineered tissues. Stem cells are widely used as seed cells in tissue engineering, and a type of pluripotent cells having self-replication ability, under certain conditions, can be differentiated into various types of cells. With the in vitro separation, culture and amplification technology of stem cells becoming mature, the research of stem cells as tissue engineering seed cells has been widely accepted, and many breakthrough scientific research progresses are achieved.

Mesenchymal stem cells are a class of pluripotent stem cells derived from early-developing mesoderm and have the potential to differentiate into dermal tissue, muscle tissue, bone and other connective tissues and circulatory systems. Taking Bone Marrow mesenchymal stem cells (BMSCs) from Bone Marrow as an example, the BMSCs can be differentiated into mesenchymal tissues such as Bone, cartilage, adipose tissue and the like under different induction conditions, and the multidirectional differentiation capability of the BMSCs, particularly the differentiation in the osteogenic direction, has unique advantages in the research of Bone tissue engineering, and can be used for culturing new Bone tissues to realize Bone injury healing. BMSCs have the advantages of convenient material acquisition and simple and convenient in-vitro separation culture method, still have differentiation capacity after being amplified for a plurality of generations, have small immunological rejection reaction after autologous BMSCs are returned to the body, are not limited by ethics, and are considered as important seed cells of bone tissue engineering.

Disclosure of Invention

In the invention, a support model simulating a cancellous bone trabecula structure is designed by a parameterized three-dimensional modeling method, so that the support model can regulate and control support parameters such as the number of micropores, the size of the micropores, the porosity, the pore wall form and the like. Beta-tricalcium phosphate powder is prepared by a hydrothermal method and is used as a main raw material, and a bionic scaffold and a grid scaffold are prepared by a 3D printing technology. The bovine femur is treated by calcining ammonium dihydrogen phosphate to prepare the cancellous bone scaffold with the remainder being high-purity beta-tricalcium phosphate. The bionic scaffold, the grid scaffold and the cancellous bone scaffold are characterized, detected and structurally analyzed, bone marrow mesenchymal stem cells are attached and osteogenic differentiation is induced, the bionic scaffold, the grid scaffold and the cancellous bone scaffold are implanted into the SD rat skull defect for 8 weeks, the in vitro biocompatibility and in vivo bone regeneration effects of the three scaffolds are evaluated, and the influence of different scaffold bionic structures on cell proliferation adhesion and in vivo bone repair effects is researched.

The research creatively constructs a novel bionic scaffold model through a 3D-Voronoi algorithm and a random distribution micropore algorithm, and applies a biological 3D printing technology to manufacture the high-precision tissue engineering scaffold. The bovine femur is calcined by using an ammonium dihydrogen phosphate secondary calcination method, inorganic components such as hydroxyapatite are removed on the basis of realizing the degreasing, decalcification and antigen removal of the allogeneic bone, the remainder is high-purity beta-tricalcium phosphate, the form of the natural cancellous bone is reserved, and the variable difference caused by the matrix material in the experiment is eliminated. Through various experiments such as representation detection, structural analysis, in vitro experiments, in vivo implantation and the like, the influence of different scaffold structures on cell proliferation adhesion and in vivo bone repair effect is comprehensively evaluated, and a reliable scaffold model and an effective detection means are provided for the bionic structure design of the bone tissue engineering scaffold.

Drawings

FIGS. 1-12D-Voronoi algorithm diagrams.

Fig. 1-2a biomimetic scaffold modeling process.

FIGS. 1-3 illustrate the structural parameter regulation of a biomimetic scaffold.

Fig. 1-4. beta. -tricalcium phosphate powder infrared absorption spectrum.

FIG. 1 is X-ray diffraction pattern of 5 beta-tricalcium phosphate powder.

FIGS. 1-6 β -tricalcium phosphate transmission electron micrographs.

FIG. 2-1 Natural cancellous bone scaffolds calcined with ammonium dihydrogen phosphate.

Fig. 2-2 three-dimensional reconstruction of a stent model.

Fig. 2-3 compressive moduli of different structural scaffolds.

FIG. 3-1 bone marrow mesenchymal stem cell culture (. times.50).

FIGS. 3-2 Giemsa staining (left:. times.100, right. times.200).

FIG. 3-3 DNA content detection.

FIGS. 3-4 detection of alkaline phosphatase Activity.

FIG. 3-5 RT-PCR detection of osteogenic differentiation related genes.

FIG. 4-1 Micro-CT scan and three-dimensional reconstruction.

FIG. 4-2 OCN immunohistochemical staining.

FIGS. 4-3 safranin fast green histological staining.

Detailed Description

The present invention will be described in further detail with reference to examples.

Example 1 design and preparation of a cancellous bone biomimetic scaffold

1.1 three-dimensional modeling of cancellous bone biomimetic scaffold

Rhinoceros is a common parameterized three-dimensional model design software, and in the research, a cancellous bone bionic model with a controllable structure is functionally designed through the Rhinoceros 5 software (v.5.1.30103.145) and a Grasshopper plug-in (v.August-27, 2014) algorithm.

The bionic support designed by the research mainly applies a three-dimensional Voronoi algorithm, a Voronoi diagram is called a Thiessen polygon, in a single plane, perpendicular lines are made at the midpoints of connecting lines of two adjacent points, and each point is divided into independent areas by the perpendicular lines. If the Voronoi algorithm is applied to a three-dimensional space, a vertical plane is made at the midpoint of a connecting line of two adjacent points, and each point is surrounded into an independent space by the vertical planes. The basic principle of the method is that a Voronoi algorithm is used for constructing a porous scaffold, micropores are formed by taking a certain number of points distributed in space as centers, and a vertical plane surrounding the micropores is subjected to morphological transformation to form a pore wall with a communicating channel (figure 1-1).

The specific modeling method is as follows: firstly, setting the shape and side length profile of a stent, so as to obtain the Total Volume (TV) of the stent, setting randomly distributed points in the stent by a Populate 3D instruction, representing Micropores (MP) in the stent, wherein the Number of the points can be increased or decreased by a Number Slider instruction (figure 1-2A); generating a number of planes around the micropores in the scaffold by Voronoi 3D commands (fig. 1-2B); assigning values to each two plane intersection divided line segments, converting the line segments into prisms with volumes (fig. 1-2C); removing plane parameters and point parameters in the stent, and reserving prisms to form a skeleton structure of the stent (fig. 1-2D); softening and softening the framework structure of the stent through a Weaverbird's Loop instruction, and increasing the internal surface area of the stent (fig. 1-2E); the scaffold model was saved and exported in STL format (fig. 1-2F).

Because the sizes, the shapes and the distribution of micropores in the bionic scaffold are irregular, the shapes and the thicknesses of pore walls are also uneven. According to the software function, the volume (BV) of the biomimetic scaffold can be calculated. Thus, we find the theoretical porosity (P) of the scaffold by the formula:

Regarding the pore size (phi) of the micropores in the scaffold, the shape of the micropores is regarded as an approximate sphere, and the pore size of 550-700um is more favorable for the proliferation and differentiation of osteoblasts according to the previous research, so the pore size is set to 600um, the regulation and control of the pore size are realized according to the regulation of the number of the micropores (NMP) in the scaffold, and the calculation refers to the following formula:

1.2 preparation of beta-tricalcium phosphate powder by hydrothermal method and preliminary characterization and detection

In the research, a hydrothermal method is adopted to synthesize beta-tricalcium phosphate, and calcium nitrate [ Ca (NO) is added according to the weight ratio of Ca/P substances of 1.5₃)₂·4H₂O]And ammonium dihydrogen phosphate [ (NH)₄)₂HPO₄]respectively dissolved in 500mL of distilled water to obtain (NH)₄)₂HPO₄The solution was slowly added dropwise to Ca (NO)₃)₂·4H₂O solution, the solution pH =6 was maintained by dropwise addition of ammonia water under a water bath at 40 ℃. And after titration, stirring for 5 hours, aging for 24 hours, filtering, washing with distilled water and ethanol respectively, and drying in a vacuum drying oven. After drying, the sample is calcined in a muffle furnace at 900 ℃ for 4 h. Grinding to obtain white beta-tricalcium phosphate powder.

1.2.1 Infrared Spectroscopy

Qualitative analysis of the composition of β -tricalcium phosphate material was performed using a fourier infrared spectrometer. Removing a small amount of beta-TCP (0.002 g) and KBr (0.15 g), mixing, grinding into uniform powder, tabletting, and scanning within the range of 4000-^-1。

1.2.2X-ray diffraction detection

The measurement was performed using an X-ray diffraction analyzer (CuK. alpha.). The energy of the electron beam is set to be 40kV, the current of the electron beam is set to be 50mA, the step width (2 theta) is 0.02 degrees, the scanning speed is 10 degrees/min, and the scanning range is 10 degrees to 80 degrees.

1.2.3 Transmission Electron microscopy

The grain size and the microscopic morphology of the beta-tricalcium phosphate are observed by a transmission electron microscope, and the operating voltage is 80 kV. And dispersing the powder sample in ethanol, and performing electron microscope analysis by sampling after ultrasonic treatment for 1 h.

1.33D printing technology preparation support

The printing preparation of the scaffolds was performed using a 3D bioplotter biological 3D printer (fig. 2-4) in this study. Before printing, the beta-tricalcium phosphate powder is ground again and fully mixed with polyvinyl alcohol solution [ omega (PVA) = 10% ] to prepare slurry. The raw material was introduced into a silo, and printing was performed at a pressure of 0.45MPa using a hyperfine nozzle (Φ = 100 μm).

Two kinds of supports are prepared by using a 3D printing technology, one kind is a bionic support STL model which is designed and derived according to the step 2.3, and model slicing and layering are carried out through software; the other is a tissue engineering scaffold with a 0/90 grid structure, which is provided with Visual mechanical software of a biological 3D printing system.

After printing, the scaffolds were removed from the platform and dried overnight at 90 ℃ and then sintered in a muffle furnace at 500 ℃ for 3h to remove the polyvinyl alcohol solvent. Randomly extracting a bracket, grinding into powder, and repeating the steps to perform infrared spectrum and X-ray diffraction detection.

1.4 results of the experiment

1.4.1 three-dimensional modeling of cancellous bone bionic scaffold

The bionic scaffold parametrically designed by utilizing the Rhinoceros software generates a porous structure similar to a cancellous bone trabecular meshwork according to a series of software algorithms, the structural parameters of the bionic scaffold can be adjusted through software instructions, and in the research, the controllable Number of Micropores (NMP) (shown in figures 1-3A), the controllable wall thickness (shown in figures 1-3B), the controllable size of micropore channels (phi channel) (shown in figures 1-3C) and the controllable scaffold shape (shown in figures 1-3D) of the bionic scaffold are realized through parameter design.

And further analyzing the volume change and the surface area change of the stent caused by the change of the structural parameters of the stent: when the parameters of the hole wall and the pore channel are not changed, the number of micropores in the bracket is increased, the volume of the bracket is reduced, the porosity is correspondingly improved, the surface area is increased, and the pore diameter is reduced; when the number of micropores in the support is constant, the pore wall is thinned, the volume of the support is reduced, the porosity is correspondingly improved, the surface area is slightly changed, and the pore diameter is correspondingly increased; when the number of micropores in the support and the shape of the pore wall are constant, the size of the micropore channel is independently changed, the size of the support is slightly increased due to the reduction of the micropore channel, the porosity is slightly reduced, the surface area of the support is obviously increased, and the influence on the pore diameter is small.

Finally, according to the previous tissue engineering scaffold structure research and the forming precision of a 3D printer, selecting a lamellar porous scaffold with the size of 2mm in height, 6mm in diameter, 360 micropores and 70% of theoretical porosity as a subsequent in vitro biocompatibility research and in vivo implantation experiment; a cubic block-shaped porous scaffold having a dimension of 10mm on a side, a number of micropores of 6364 each, and a theoretical porosity of 70% was selected for scaffold structural analysis.

1.5 Infrared Spectroscopy

The infrared absorption spectrum of the white powder obtained by the hydrothermal method is shown in FIGS. 1 to 4.

From the analysis result of the infrared spectrum, 1042cm^-1、603cm^-1、579cm^-1Wave number of (d) corresponds to the characteristic peak of phosphate group, 3420cm^-1The functional group has better consistency with the standard spectrogram of the infrared spectrum of the beta-tricalcium phosphate.

1.6.1X-ray diffraction detection

The X-ray diffraction measurement results are shown in FIGS. 1 to 5.

The characteristic main peak 2 theta =31.0 degrees, the secondary strong peak 2 theta =27.8 degrees, 34.5 degrees and 16.9 degrees of the sample map are all matched with the characteristic peak of a standard comparison card (JCPDS 09-0169), and the generated product is beta-tricalcium phosphate.

1.6.2 Transmission Electron microscopy

The results of transmission electron microscope observation are shown in FIGS. 1 to 6.

The observation result of the transmission electron microscope of the beta-tricalcium phosphate shows that the grain diameter of the beta-tricalcium phosphate is nano-scale, the shape is regular, the size is uniform, the grain diameter range of the crystal is 50-300nm, and the split grains show a small amount of agglomeration phenomenon.

1.6.33D printing and preparing bionic bracket

Beta-tricalcium phosphate powder is mixed with polyvinyl alcohol to form slurry, and the slurry is made into a slice bracket with the height of 2mm and the diameter of 6mm and a cubic square bracket with the side length of 10mm by a biological 3D printer. The 3D printing technology well keeps the complex three-dimensional structure of the model, the printed support has a through porous structure, and meanwhile, the bionic support and the grid support both keep the complex geometric structure of the porous support. The printed bracket is taken down from the working platform and is put into a drying oven to be dried overnight at 90 ℃, the original structure of the bracket is still kept after the drying, and the bracket does not generate larger volume and form changes and does not crack or damage. After the randomly extracted bracket is ground into powder, infrared spectrum and X-ray diffraction detection show that the bracket material is only beta-tricalcium phosphate and polyvinyl alcohol is completely removed.

1.7 summary

In this chapter, a spongy bone form porous bionic scaffold capable of controlling a scaffold parameter structure is designed through parametric three-dimensional modeling, and according to the previous research on the tissue engineering bionic scaffold structure, the pore size of 600um and the higher porosity of 70% are selected for subsequent research. And beta-tricalcium phosphate prepared by a hydrothermal method is used as a raw material, and the spongy bone bionic scaffold and the 0 DEG/90 DEG grid scaffold are finally prepared by performing layer-by-layer printing and layer-by-layer superposition preparation molding on the crystal form by using a pneumatic extrusion type high-precision 3D printing method. In order to determine that the powder prepared by the hydrothermal method and the dried stent are beta-tricalcium phosphate materials, infrared spectroscopy, X-ray diffraction and transmission electron microscope detection are carried out on the materials, and the results show that the stent material is nano-beta-tricalcium phosphate, and can be well applied to meet the requirements of bone tissue engineering materials.

Example 2 ammonium dihydrogen phosphate method for calcining natural bovine cancellous bone and detection of scaffold Structure

2.1 ammonium dihydrogen phosphate calcination of bovine cancellous bone

2.1.1 bone preparation and pretreatment

The method comprises the steps of purchasing fresh adult bovine femurs from local markets, sawing the femoral necks above and the inner and outer condyles from the two ends of the femurs by using a steel saw, soaking the fresh adult bovine femurs for 3 hours by using 0.5M/L sodium hydroxide at normal temperature and normal pressure, boiling the fresh adult bovine femurs for 30 minutes, sawing the fresh adult bovine femurs into spongy bone blocks with the sizes of 10 multiplied by 10mm by using the steel saw, soaking the spongy bone blocks for 24 hours by using 0.5M/L sodium hydroxide, and drying.

2.1.2 calcining of bone pieces by ammonium dihydrogen phosphate method

Placing the bone blocks in a muffle furnace, slowly heating (6 ℃/min) to 900 ℃, maintaining for 4h, cooling and taking out. Soaking in 0.5M ammonium dihydrogen phosphate for 24 hr, oven drying, placing in a muffle furnace, slowly heating (6 deg.C/min) to 1000 deg.C, and maintaining for 4 hr. And cooling, taking out, extracting a calcined bone, grinding into powder, and performing subsequent material characterization detection.

2.1.3 Infrared Spectroscopy

Qualitative analysis of calcined bone material composition using fourierAnd detecting by an infrared spectrometer. Removing a small amount of beta-TCP (0.002 g) and KBr (0.15 g), mixing, grinding into uniform powder, tabletting, and scanning within the range of 4000-^-1。

2.1.4X-ray diffraction detection

The calcined bone component was measured using an X-ray diffraction analyzer (CuK α). The electron beam energy was set to 40kV, the electron beam current was set to 50mA, the step width (2. theta.) was 0.02 DEG, and the scanning speed was 10 DEG/min.

2.2 Stent Structure detection

2.2.1 Micro-CT scanning and three-dimensional reconstruction

And scanning the cancellous bone bionic support prepared by 3D printing, the 0 degree/90 degree grid support and the calcined bone support by using a Micro-CT (Micro-computed tomography), wherein the operating voltage of the Micro-CT is set to be 80kV, the current is 200mA, and the scanning layer is 48 microns thick. After scanning, introducing a CT image by using a Mimics (v 17.0) software to reconstruct a three-dimensional image.

2.2.2 scanning Electron microscopy

The microstructures of three scaffolds with different structures were observed using a scanning electron microscope. And (5) spraying gold after drying the support in vacuum, and setting the operating voltage to be 15 kV.

2.2.3 mechanical Property testing

Three supports with different structures are placed between two loading heads of the universal material testing machine, a 10KN sensor is used, the loading speed is 1.0mm/min, and the loading is stopped when the supports are compressed by 3.0mm, so that the stress-strain test result is obtained. The compressive modulus was determined by fitting the slope of the stress-strain curve (initial 5%).

2.3 results of the experiment

The cancellous bone pieces after the secondary calcination of the ammonium dihydrogen phosphate are white, and a large number of irregular porous cavities can be seen by naked eyes, so that the bovine femoral bone treated by the calcination of the ammonium dihydrogen phosphate is prompted to retain the porous structure of the cancellous bone (figure 2-1).

The infrared spectrum and the X-ray diffraction result show that the residue of the natural cancellous bone calcined by the ammonium dihydrogen phosphate method is high-purity beta-tricalcium phosphate by comparing the infrared characteristic peak of the beta-tricalcium phosphate with the X-ray diffraction standard comparison card.

The scaffold model (figure 2-2) which is scanned by Micro-CT and reconstructed in three dimensions is cut into the size of 10 multiplied by 10mm by Rhinoceros 5 software, the volume, the surface area, the micropore communication rate and other structural parameters of the scaffold are analyzed by the software, and the analysis results are shown in tables 2-3.

Table 2-3 comparison of scaffold geometry parameters (n =20, ± S)

Table.3-3 Comparison of the geometric structure parameters of the scaffolds（n=20，±S）

Item	Bionic support	Grid support	Calcined bone
				Porosity (%)	68.60±3.16	53.01±2.26	66.53±13.07
Surface area (mm)²）	5910.47±501.26	4614.10±253.88	7521.41±1428.70
				Micropore communication ratio (%)	97.52±2.33	100±0.00	92.77±3.09
Pore size (mum)	535.2±113.51	836.11±37.01	339.40±59.72
				Shape of micro-pores	Irregular spherical shape	Cylindrical shape	Irregular lacuna

The bionic scaffold comprises a cancellous bone bionic scaffold, a 0 degree/90 degree grid scaffold and a calcined bone scaffold, wherein three scaffolds with different structures show different structural parameters under the same size: the porosity of the bionic scaffold is 68.60 +/-3.16%, the theoretical porosity of the composite parametric design is 70%, and the porosity is similar to the porosity of 66.53 +/-13.07% of the calcined bone scaffold, and the difference of the two groups has no statistical significance (p = 0.35)>0.05), the porosity of the lattice scaffold is low, 53.01 + -2.26%, and the difference between the lattice scaffold and the other two groups is statistically significant (p)<0.05); calcined bone scaffolds surface area 7521.41 + -1428.70 mm²The largest of the three groups, the surface area of the bionic scaffold is 5910.47 +/-501.26 mm²The surface area of the grid support is the minimum and is 4614.10 +/-253.88 mm²(ii) a The micropore communication rate is analyzed by Boolean operation with a cubic entity with the size of 10 multiplied by 10mm, the micropore communication rate of the grid support is 100%, the bionic support is 97.52 +/-2.33%, and the calcined bone support is 92.77 +/-3.09%.

According to the structural analysis of a three-dimensional reconstructed support model of Micro-CT data, the bionic support and the calcined bone support have higher porosity and surface area, the higher porosity means that more space exists in the support in unit volume, so that the support is beneficial to loading a large amount of cells, extracellular matrix and growth factors, and the larger surface area is larger for the cells to proliferate and adhere; the porosity and the surface area of the grid support are low, but the micropore communication rate is 100 percent, namely, the support is not provided with a closed cavity, and the growth of cells in the support, the transportation of nutrient substances and the discharge of wastes are facilitated. However, the micropore communication rate of the bionic support does not reach 100%, and the micropore communication rate of the bionic support is not consistent with the structure of a parameterized model without a closed cavity, and the bionic support is considered to be more precise and complex than a grid support, so that a small amount of closed cavities exist in a small amount of supports due to material collapse, uneven material extrusion and fine deformation generated during support sintering in the 3D printing and forming process. And the grid support is simple in structure, so that the situation that the support is influenced by gravity to cause material collapse is not obvious. The reason why the closed cavity exists in the calcined bone scaffold is considered to be that the inorganic components of the bone tissue are also damaged to a certain extent because the organic substance is sintered during the calcination process, thereby generating the closed cavity. Secondly, after the model is built by the Mimics software, the internal structure of the calcined bone scaffold is complex, the reconstructed three-dimensional model is subjected to large-area model fusion, and part of adjacent trabeculae cannot be distinguished, so that a closed cavity in the three-dimensional model is formed.

Different support structures have certain influence on the mechanical properties, and the compression test results of the three groups of supports are shown in figures 2-3. Compared with a bionic support and a cancellous bone support, the grid structure has a higher compression modulus of 19.83 +/-0.37 MPa, the bionic support has a compression modulus of 12.60 +/-4.05 MPa, and the calcined bone support has a lower compression modulus of 6.28 +/-1.19 MPa.

The material fiber of the grid structure is regular and compact in trend, the bearing capacity shown in a compression test is stronger, and according to the comparison of structural parameters, the porosity of the grid support under the same volume is lower, so that more materials are contained, and the modulus is higher in the compression test. The bionic scaffold and the calcined bone scaffold show lower compression modulus in a compression test because of disordered and irregular material fibers and higher porosity, and the reason that the compression modulus of the calcined bone scaffold is low is considered, and the mechanical property is poorer because the organic matter is possibly caused to flow by calcination, so that the bone tissue is partially loosened and lost. On the other hand, the original mechanical properties of the beta-tricalcium phosphate biological ceramics are poor, so that the three beta-tricalcium phosphate material scaffolds show no appreciable mechanical properties.

2.4 summary

The content of the chapter is that the bovine femur is treated by an ammonium dihydrogen phosphate secondary calcination method, and the high-purity beta-tricalcium phosphate natural cancellous bone scaffold is obtained through material characterization and detection, which proves that the ammonium dihydrogen phosphate secondary calcination method can be used as a mature experimental method for preparing the beta-tricalcium phosphate natural cancellous bone scaffold except the bovine femur. And the support materials of the bionic support and the grid support prepared in chapter 2 are unified, irrelevant variables are eliminated, and the framework parameters and the mechanical properties of the support are researched through Micro-CT scanning, three-dimensional reconstruction and compression resistance test based on the research and analysis.

The research in this chapter shows that the spongy bone bionic scaffold and the calcined bone scaffold both have higher porosity and surface area, and are superior to the grid scaffold structure used in the traditional tissue engineering, which indicates that the spongy bone bionic scaffold is similar to the natural spongy bone tissue in structural parameters, and although there is a certain difference in form, the outstanding structural characteristics of the spongy bone bionic scaffold can provide theoretical basis for the use of the spongy bone bionic scaffold as a tissue engineering scaffold model.

Example 3 in vitro biological evaluation

3.1 extraction and culture of rat bone marrow mesenchymal Stem cells

Selecting 1 male SD rat of 4 weeks old, killing by cervical dislocation method, soaking in 0.5% iodophor for 3min for sterilization, separating femur and tibia at two sides under aseptic condition, flushing out bone marrow with low-sugar DMEM culture solution, and centrifuging the obtained cell suspension at 2000r/min for 10 min. Then re-suspended with 15mL DMEM containing 10% fetal bovine serum and 1% double antibody at 1X 10⁸L^-1Inoculating at a density of 25cm²Culturing in a culture flask at 37 deg.C in a CO2 constant temperature incubator. The first half-change was done 24h after seeding to remove non-adherent cells. After that, the total amount of the solution was changed 1 time every 3 d. When the growth and fusion of adherent cells reach more than 90%, digesting and subculturing by using 0.25% trypsin, and subculturing according to the proportion of 1: 3.

3.2 Giemsa staining

Dynamically observing the morphological change of the mesenchymal stem cells in the growth process from the primary culture under an inverted phase contrast microscope andAnd (6) taking a picture. Adjusting the cell concentration of the 2 nd generation cells to 5X 10⁷L^-1And plated into 24-well cell culture plates, 0.75mL per well. When the cells are uniformly distributed and about 90% of the cells are fused, washing the cells for 3 times by using a phosphate buffer solution, fixing the cells by using methanol for 10min, dyeing the cells by using Giemsa dye liquor for 2min, washing the cells by using distilled water, and observing and taking pictures under a microscope.

3.3 cell inoculation on scaffolds for Co-culture

Soaking three scaffolds with different structures in 70% ethanol for 2h, removing ethanol, washing with alkaline phosphatase buffer solution, sterilizing under ultraviolet irradiation for 30min, transferring to 24-well tissue culture plate, soaking in DMEM culture solution containing 10% fetal calf serum by volume fraction for 24h, and adjusting cell concentration of 2 nd generation cells to 5 × 10⁷L^-1The cells were inoculated into 24-well cell culture plates, 1mL of the cells were inoculated into each well, and after incubation for 2 hours, 1mL of the whole culture was added to each well.

3.4 DNA content determination

After 1, 7, 14 and 21 days of inoculation into the scaffolds, cell proliferation assays were performed by measuring the DNA content of each sample according to the PicoGreen dsDNA assay kit instructions. A24-well plate containing the samples was digested with 5mM EDTA, 5mM L-cysteine, 0.1M phosphate buffer, and papain (400 lg/mL) at pH6. Add test buffer to make up to 100. mu.L and add 100. mu.L of PicoGreen reaction reagent to each well. And detecting the light absorption value by using a multifunctional microplate reader, and exciting by adopting double fluorescence excitation with the excitation wavelength of 460nm at 360 nm. And calculating the DNA content of the sample by contrasting with the standard DNA content curve.

3.5 live/dead cell staining

After 1, 7, 14 and 21 days of seeding onto the scaffold, the medium was removed, washed twice with phosphate buffer, 0.25mL of stain (1 mM Live-Dye and 205mg/mL propidium iodide) was added per well, and incubated for 20 minutes at 37 ℃ in a carbon dioxide incubator. The staining agent was removed, washed thoroughly with phosphate buffer and observed under an inverted fluorescence microscope.

3.6 osteogenic Induction of differentiation

The SD rat bone marrow mesenchymal stem cells are digested with pancreatin when being cultured to the fourth generation, and the digestion is carried out at 5X 10⁷L^-1The cells were seeded onto each of the scaffolds, and transferred to a 37 ℃ carbon dioxide incubator for 3 hours to allow the cells to adhere to the porous scaffolds. After 3h, 1.0 mL of DMEM complete growth medium containing 10% fetal bovine serum and 1% double antibody was added to each well and the culture was continued in a 37 ℃ carbon dioxide incubator. After 24h the DMEM complete growth medium was changed to DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin double antibody, as well as dexamethasone at a concentration of 100 nM, vitamin C at 10. mu.g/mL, sodium beta-glycerophosphate at 10 mM.

3.7 alkaline phosphatase Activity detection

Alkaline phosphatase activity was measured by the p-nitrophenyl phosphate method 1, 7, 14 and 21 days after osteogenic induction. Removing the culture medium in the well plate, washing with phosphate buffer solution for 2 times, taking out the sample, transferring to a 2mL centrifuge tube, adding p-nitrophenol solution, lysing at 37 ℃ for 2h, measuring the absorbance at 405 nm, and calculating the alkaline phosphatase activity by referring to an ALP standard curve.

3.8 detection of osteogenesis-related Gene expression

After 1, 7, 14 and 21 days of osteogenic induction, the levels of osteogenic-related genes expressed by the cell-scaffolds, including transcription factor 2 (Runx 2), Osteocalcin (OCN), Osteopontin (OPN), type I collagen (COL 1a 1), were determined. Removing culture medium from the well plate, washing with phosphate buffer solution for 2 times, freezing with liquid nitrogen, grinding the sample into powder, extracting total cellular RNA with TaKaRa MiniBEST RNA extraction kit, and using PrimeScript^TMcDNA synthesis was performed using RT Master Mix kit. After the reverse transcription is finished, a specific osteogenesis related gene primer is added into a real-time fluorescence quantitative PCR analyzer, the expression (GAPDH) of glyceraldehyde-3-phosphate dehydrogenase is used as an endogenously controlled housekeeping gene, and 2 is utilized^−ΔΔCTThe expression of the relevant gene is calculated. The primer list is shown in Table 3-2.

TABLE 3-2 qRT-PCR primer Table

3.9 statistical methods

Statistical treatment was performed using SPSS (v 17.0) with P <0.05 as a significance criterion. (. P <0.05, P < 0.01)

3.10 results of the experiment

Cells washed and separated from the marrow cavity of an SD rat are cultured for 24 hours and 48 hours after the first passage and observed under an inverted phase contrast microscope, a small amount of cells grow in an adherent manner, the size is uniform, the shape is a long fusiform shape, and the cells grow in a fibroblast-like colony manner after being cultured for 4 days. The image of the cell culture at some time points is shown in FIG. 3-1.

After 1 day of culture, cells were Giemsa stained as shown in FIGS. 3-2, with purple-red nuclei and bluish-purple cytoplasm and nucleoli.

The DNA content test results are shown in FIGS. 3-3. The proliferation condition of the bone marrow mesenchymal stem cells on different structural scaffolds is determined by DNA content detection, and the result shows that after 21 days of culture process, the DNA content shown on the three groups of scaffolds shows a continuous rising trend along with the time. Wherein the content of DNA on the bionic scaffold is much higher than that on the grid scaffold and the calcined bone scaffold at 14 days and 21 days. The grid scaffold shows higher DNA content in 1 and 7 days, which indicates that the grid scaffold has promotion effect on early cell adhesion and shows higher initial adhesion rate. Cells on the calcined bone scaffold showed lower DNA content in 1 and 7 days, considering that since the scaffold had a larger surface area and the initial density of adherent cells was higher, some cells showed apoptosis after being seeded on the scaffold, showing lower DNA content in 1 and 7 days and significantly increased DNA content in 14 and 21 days.

The survival condition of the bone marrow mesenchymal stem cells on different structural scaffolds is determined by live/dead cell staining, and the results show that the bone marrow mesenchymal stem cells of SD rats on different structural scaffolds have different cell adhesion effects. At different time points, the results of cell adhesion quantity and DNA content are similar, and the experimental result shows that the mesenchymal stem cells fall off and are washed and removed because the mesenchymal stem cells cannot adhere to the scaffold after apoptosis on the scaffold. With the increase of the culture time, the mesenchymal stem cells gradually transfer from the adhesion on the surface of the scaffold to the inside of the scaffold, and the cell adhesion quantity on the surface of the scaffold is more than that on the inside. Experiments show that the scaffolds with three structures are all suitable for the adhesion and migration of bone marrow mesenchymal stem cells.

The results of alkaline phosphatase activity are shown in FIGS. 3 to 4. Bone marrow mesenchymal stem cells are subjected to osteogenic induction, and the osteogenic differentiation effect is detected by measuring the activity of alkaline phosphatase. The results show that the alkaline phosphatase activity undergoes a first-to-last change within 21 days, and after 14 days of culture on scaffolds of different structures, the alkaline phosphatase activity reaches a maximum, wherein the alkaline phosphatase activity expressed on calcined bone scaffolds is the greatest, the biomimetic scaffolds are the next to the biomimetic scaffolds, and the lattice scaffolds are the smallest. After further culturing for 21 days, the alkaline phosphatase activity was decreased.

The results of the osteogenesis-related gene expression are shown in FIGS. 3 to 5. Bone marrow mesenchymal stem cells are subjected to osteogenesis induction, and the osteogenic differentiation effect is detected by detecting the expression level changes of osteogenesis related gene transcription factor 2 (Runx 2), Osteocalcin (OCN), Osteopontin (OPN) and type I collagen (COL 1A 1). The expression of Runx2 experienced a first-to-last-to-fall trend on three different scaffolds, reaching a peak at 7 days and finally declining gradually. The expression of OCN is subjected to the trend of first rising and then falling on three scaffolds with different structures, the expression reaches the highest value after 14 days of culture, and the expression of OCN of the bionic scaffold is two times higher than that of a calcined bone scaffold and a grid scaffold. On the grid support and the calcined bone support, the expression level of OPN shows a gradual rising trend, and reaches a maximum value 21 days after culture, while the expression level of OPN of the bionic support shows a rising-before-falling trend, reaches the maximum value 14 days, and is reduced 21 days, and the expression level of OPN in the bionic support is higher than that of the calcined bone support and the grid support at time points of 1, 7 and 14 days. The expression of COL1a1 experienced a gradual increase in the expression level on all three differently configured scaffolds, with the calcined bone scaffold exhibiting slightly higher expression levels than the biomimetic scaffold, significantly higher than the lattice scaffold.

3.11 knots

In the research of this chapter, the bone marrow mesenchymal stem cells of SD rats are separated and extracted, and are cultured and identified in vitro, and experiments show that the extracted bone marrow mesenchymal stem cells have good growth, vigorous proliferation and osteogenic differentiation potential. After being inoculated on three scaffolds with different structures, the scaffolds are subjected to osteogenic induced differentiation, and after 21 days of in-vitro co-culture, the three scaffolds show good cell biocompatibility, and in a preliminary in-vitro osteogenic induced differentiation experiment, the scaffolds with different structures show osteogenic differentiation potentials of different osteogenesis bones, specifically, the bionic scaffold shows more excellent osteogenic differentiation potentials of the mesenchymal stem cells than a calcined scaffold and a grid scaffold.

Example 4 animal experiments

4.1 animal Experimental grouping and modeling

The animal experimental study has been approved by the southern medical university laboratory animal center, 16 10-week-old Sprague-Dawley (SD) male rats are selected as experimental animals for the experiment, a circular defect with the diameter of 6mm is drilled in the central area of the skull by a trephine with the diameter of 6mm after anesthesia, the thickness of the drilled skull is about 2mm, a sheet-shaped bionic scaffold and a grid scaffold which are prepared in chapter 2 and have the height of 2mm and the diameter of 6mm are co-cultured with bone mesenchymal cells and are milled and cut into the same size, and the calcined bone scaffold and the bone mesenchymal cells are induced to have osteogenic differentiation for implantation.

16 rats were divided into 4 groups, each group consisting of 4 rats, including a biomimetic scaffold group, a mesh scaffold group, a calcined bone scaffold group, and a blank control group. The first three groups are respectively implanted with a bionic scaffold, a grid scaffold and a calcined bone scaffold which are loaded with cells, and the fourth group is only sutured after the bone defect is manufactured and is not implanted with materials. Rats were sacrificed for further study 4 weeks and 8 weeks after implantation into the rats.

4.2 animal experiments on skull defect repair

Before anesthesia, the weight is measured, and 75mg/kg ketamine and 10mg/kg xylazine are administered for intraperitoneal injection anesthesia. The anesthesia and replacement skin is disinfected, the operation is carried out under the aseptic condition, the skin is cut along the midline of the skull, and the skin is pulled to expose the visual field. A6 mm diameter replacement drill was used to drill a hole in the central region of the skull, resulting in a 6mm circular skull defect. Removing the defective skull by using sterile normal saline for fixing and washing, and checking the edge of the defective area to determine that no residual bone exists. The bracket is placed into the hard meninges according to groups for filling and repairing, and the bracket is placed to be in contact with the hard meninges, so that soft tissues and skin are prevented from being sutured after the completion of the repair. And (5) feeding in cages after operation.

At two time points of 4 weeks and 8 weeks, 2 rats per group were selected each time, sacrificed by anesthesia, and fixed with 10% paraformaldehyde at 4 ℃ for further study.

4.3 Micro-CT analysis

The experimental animal is horizontally placed on a sample rack for Micro-CT scanning, and the scanning parameters are as follows: the voltage is 59kV, the current is 167mA, the scanning layer is 18um thick, and after scanning, the bone defect area is selected to be subjected to bone mineralization density analysis by using software for Micro-CT bone analysis. And importing the Micro-CT image data into the Mimics (v 17.0) software to reconstruct the three-dimensional image.

4.4 immunohistochemical analysis

After the rat skull was fixed with 4% paraformaldehyde for 24 hours, decalcification was performed with 10% EDTA at 4 ℃, dehydration was performed with gradient alcohol, wax dipping and embedding were performed, longitudinal cutting was performed from the proximal to the distal in the center of the bone defect region, 10 sections per sample were performed, and tissue staining and immunohistochemical staining were performed.

4.5.1H & E staining

The dewaxed sample slice was stained in hematoxylin stain for 8 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for several seconds, washed with tap water, rewetted with 0.6% ammonia water, and washed with running water. The sections were stained in eosin stain for 2 min. And dehydrating the slices with 95% ethanol and anhydrous ethanol for 3min respectively, soaking in xylene I, II for 5min respectively, air drying, sealing with neutral gum, observing and photographing under microscope, collecting image information, and analyzing.

4.5.2 OCN staining

The dewaxed sample sections were incubated with proteinase K solution at 37 ℃ for 10 minutes, washed 3 times with triethanolamine-buffered saline solution (Tris-HCl buffered saline solution, pH 7.4) at pH 7.4, and washed 2 times with phosphate buffer containing 0.1% Triton X-100. Blocking with triethanolamine buffered saline containing 1% bovine serum albumin for 1h, and washing with triethanolamine buffered saline. OCN primary antibody was added at a concentration of 10. mu.g/mL and incubated at 4 ℃ for 12 h. Washing with triethanolamine buffered saline solution containing 0.2% Tween 20 for 3 times, adding OCN secondary antibody with concentration of 5 μ g/mL, incubating at room temperature for 2h, washing with triethanolamine buffered saline solution containing 0.2% Tween 20 for 3 times, adding DAPI with concentration of 1.0 μ g/mL, and incubating at room temperature for 3 min. After being washed by triethanolamine buffer saline solution, the solution is observed and photographed under a microscope, and image information is collected and analyzed.

4.5.3 safranin-fast Green staining

The dewaxed sample slice is placed into hematoxylin staining solution for staining for 3min and then washed with tap water, then washed with 1% hydrochloric acid alcohol (prepared by 70% ethanol) after color separation for 15s, placed into 0.02% fast green staining solution for staining for 3min, then washed with 1% glacial acetic acid to remove residual fast green, and placed into 0.1% safranin O staining solution for staining for 3 min. Dehydrating with 95% ethanol and anhydrous ethanol for 3min, soaking in xylene I, II, drying in the air after 5min, sealing with neutral gum, observing and photographing under microscope, collecting image information, and analyzing.

4.5.4 CD31 staining

deparaffinized sections of the samples were stained for CD 31-specific antigen. Working solution was prepared by diluting CD31 primary antibody at a ratio of 1: 100. Inactivating endogenous peroxidase with 3% hydrogen peroxide, and microwave heating and repairing with 0.01M citrate buffer solution (pH6.0); blocking with triethanolamine buffered saline containing 1% bovine serum albumin for 1h, and adding CD31 primary antibody working solution dropwise to incubate at 4 deg.C for 12 h. And (4) returning to room temperature, washing with a phosphate buffer solution, dropwise adding corresponding secondary antibody, and performing DAB color development. And observing and photographing under a microscope, and acquiring and analyzing image information.

4.6 results of the experiment

The anesthesia and the operation are carried out smoothly, the edge of the drilled hole is smooth and regular, the defect skull is removed completely, the dura mater is intact during the operation, and the brain tissue is not damaged. After operation, the fish is fed in cages, and the feeding nursing is given to relieve pain and resist infection.

The results of the Micro-CT scan and the Mimics reconstruction are shown in FIG. 4-1. New bone tissues are formed on the three different-structure scaffolds after being implanted into SD rat skull defects for 4 weeks, the new bone tissues can grow along porous pores of the scaffolds, more new bone tissues grow on the bionic scaffolds and the calcined scaffolds, and no new bone tissue grows on the blank control group defect region and the defect region edge. The new bone tissue growth volume is larger after 8 weeks of implantation SD rat skull defect, the new bone tissue of the bionic scaffold group almost fully expands the whole scaffold pore, the new bone growth volume of the calcined bone scaffold group is inferior, the new bone growth volume of the grid scaffold group is inferior, and the blank control group still has no new bone tissue growth.

The analysis result of the bone mineralization density of the new bone growth part with bone defect shows that the bone mineralization density at the boundary line of the bionic bracket is higher, which shows that the bionic bracket structure has the effect of forming the new bone and shows better bone repair effect.

H & E staining was used to detect the formation of fibroblasts, chondrocytes and reticulated original bone and their respective morphologies. The staining results showed that new bone tissue was stained deep pink, hypertrophic chondrocytes were stained purple, and fibroblasts were stained brown.

OCN staining results are shown in FIG. 4-2. The cytoplasm shows a positive reaction when the staining is positive, the distribution of the brown yellow particles can be seen, the periphery of the cell nucleus is heavily stained, part of the cell nucleus is covered by the brown yellow particles, and other cells can see blue cell nuclei.

The results of safranin fast green staining are shown in FIGS. 4-3. The distribution of green-stained bone tissue and red-stained cartilage tissue, and the arrangement disorder of new bone and cartilage tissue can be seen. The blank control group had no positive staining, only mild inflammatory cells, and no bone and cartilage tissue repair.

The CD31 staining result shows that the newborn bone tissue has a small amount of positively stained vascular endothelial cells arranged in a ring shape, and platelets in the lumen also show positive staining

4.8 Small knot

The content in this chapter is through animal implantation experiment, utilize three-dimensional reconstruction of imaging science and histology, immunohistochemical staining, the influence of different structural support to the osteogenesis effect in vivo of analysis. Analysis results of Micro-CT and three-dimensional reconstruction show that the scaffolds with three structures show good osteogenesis effects along with the time extension, wherein the osteogenesis effects of the bionic scaffold are optimal, and the inside of the scaffold in the bone defect region is almost full of new bone tissues at week 8, so that the three-dimensional scaffold structure of the bionic scaffold is proved to be favorable for guiding the growth of the bone tissues after the scaffold is implanted into the bone defect region.

Similarly, immunohistochemistry and histological staining also demonstrated the excellent in vivo osteogenic effect of the biomimetic scaffolds. And through CD31 immunohistochemical staining, generation of vascular tissues in the bionic scaffold can be seen, effective vascularization of tissue engineering bone is realized, and possibility is provided for stable growth of new bone tissues, gradual metabolism of scaffold materials and material exchange of surrounding tissues.

Claims

1. The invention creatively constructs a novel bionic scaffold model through a 3D-Voronoi algorithm and a random distribution micropore algorithm, and adopts a biological 3D printing technology to prepare the high-precision bone tissue engineering scaffold by taking beta-tricalcium phosphate powder prepared by a hydrothermal method as a raw material.

2. the biomimetic scaffold of claim 1 may be used in tissue engineered bone or components.

3. Use of the tissue engineering scaffold of claim 1 for the clinical repair of various bone defects.

4. The biomimetic scaffold of claim 1 is a composite scaffold of cell-inorganic material, characterized in that the scaffold comprises the tissue engineering scaffold of claim 1 and any other kind of seed cells.