阅读说明：本技术 一种3d打印技术制备的松质骨仿生支架的设计与应用 (Design and application of cancellous bone bionic scaffold prepared by 3d printing technology ) 是由 孙晗笑 利时雨 于 2018-06-06 设计创作，主要内容包括：本发明通过3D-Voronoi算法和随机分布微孔算法构建了新型仿生支架模型,并运用生物3D打印技术制作了高精度的该组织工程支架。运用磷酸二氢铵二次煅烧法煅烧牛股骨,在实现异体骨脱脂脱钙去抗原的基础上,去除了羟基磷灰石等无机成分,使余物为高纯度的β-磷酸三钙,保留了天然松质骨形态的同时消除了实验中因基质材料带来的变量差异。通过表征检测、结构分析、体外实验、体内植入等多种实验,全面地评价不同支架结构对细胞增殖粘附和体内骨修复效果的影响,为骨组织工程支架的仿生结构设计提供了可靠支架模型和有效检测手段,可用于临床各种骨缺损的使用。(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.)

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.

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.