阅读说明：本技术 自体成骨细胞3d生物复合打印颅骨骨瓣修复新西兰兔颅骨缺损模型的方法 (Method for repairing skull defect model of New Zealand rabbit by self osteoblast 3D biological composite printing skull flap ) 是由 孙鹏 邓文帅 董作祥 于 2020-04-24 设计创作，主要内容包括：本发明公开了自体成骨细胞3D生物复合打印颅骨骨瓣修复新西兰兔颅骨缺损模型的方法,属于颅骨修复技术领域。该自体成骨细胞3D生物复合打印颅骨骨瓣修复新西兰兔颅骨缺损模型的方法,使打印的颅骨瓣精密个体化,达到颅骨力学要求,同时使细胞在支架立体空间中均匀分布,维持细胞正常营养及代谢,保证细胞活性,将打印好的仿真颅骨瓣即时移植修复新西兰兔颅骨缺损模型,应用组织病理学、细胞免疫学及影像学等方法检测成骨细胞活性、支架结构变化、颅骨瓣与骨缘融合情况等,目的为颅骨缺损修补探索一种全新的生物材料,为临床颅骨缺损修补建立新的治疗模式,同时也为将来3D生物多材料、多细胞的机体器官打印提供重要的实验基础和理论依据。(The invention discloses a method for repairing a new zealand rabbit skull defect model by using an autologous osteoblast 3D biological composite printing skull flap, and belongs to the technical field of skull repair. The method for repairing the skull defect model of the New Zealand rabbit by the autologous osteoblast 3D biological composite printing skull flap has the advantages that the printed skull flap is precisely individualized to meet the mechanical requirements of the skull, the cells are uniformly distributed in the three-dimensional space of the bracket, the normal nutrition and metabolism of the cells are maintained, the activity of the cells is ensured, the printed simulated skull flap is immediately transplanted to repair the skull defect model of the New Zealand rabbit, the methods of histopathology, cellular immunology, imaging and the like are applied to detect the osteoblast activity, the structural change of the bracket, the fusion condition of the skull flap and the bone margin and the like, the aim is to explore a brand new biomaterial for repairing the skull defect, a new treatment mode is established for clinical skull defect repair, and important experimental basis and theoretical basis are provided for printing of organism organs of 3D biological multi-materials and multi-cells in the future.)

1. The method for repairing the skull defect model of the New Zealand rabbit by the autogenous osteoblast 3D biological composite printing skull flap is characterized in that: the method for preparing the raw materials comprises the following steps:

s1, firstly, taking out autogenous bone from a New Zealand rabbit, separating and culturing osteoblasts as seed cells for later use, and mixing hydroxyapatite powder and polycaprolactone according to a mass ratio of 1.2: 1, and fully mixing to obtain the pretreated 3D printing powder material.

And S2, fully mixing the 3D printing powder material prepared and pretreated in the above step with a phosphoric acid solution to prepare the pretreated 3D printing mixed material.

S3, mixing the prepared pre-processed 3D printing powder material with the pre-processed 3D printing mixed material through a phosphoric acid solution, and storing the mixture in an environment of 18-25 ℃ below zero for standby use to obtain the 3D printing initial material.

2. The method for repairing the skull defect model of the New Zealand rabbit by the autologous osteoblast 3D biological composite printing skull bone flap according to claim 1, wherein the method comprises the following steps: the 3D model is established by the following steps:

and S1, performing solid scanning on the defected diseased part of the head of the New Zealand rabbit by adopting a 3D scanner to obtain three-dimensional data of the head image of the New Zealand rabbit and recording the three-dimensional data.

And S2, secondly, carrying out 3D modeling by adopting modeling software, importing the three-dimensional data of the head image of the New Zealand rabbit in S1 into the modeling software, and processing the data by the modeling software to obtain a 3D stereo model diagram.

3. The method for repairing the skull defect model of the New Zealand rabbit by the autologous osteoblast 3D biological composite printing skull bone flap according to claim 1, wherein the method comprises the following steps: the 3D printing steps are as follows:

s1, when seed cells are implanted into the 3D printing initial material, the seed cells are printed into a three-dimensional structure, the autologous osteoblasts of the New Zealand rabbit are respectively mixed with agarose, methylcellulose or alginate and a thermal reversible gel material, the cell suspension can be accurately positioned and printed on the surface of the thermal reversible gel through a printer, after the cells are printed on one layer of gel, a new layer of material is covered and solidified, and the process is repeated continuously to obtain a three-dimensional cell structure, namely a cell matrix material mixture.

And S2, introducing the three-dimensional data of the 3D stereoscopic model into a 3D printing device, introducing the stored 3D printing initial material and the cell matrix material mixture into the 3D printing device, and performing printing operation in an environment at minus 25 ℃.

And S3, in the 3D printing process, printing the cell matrix material mixture layer by layer according to a pre-designed model under the control of a computer to form a specific three-dimensional structure, and chemically crosslinking the cell matrix material mixture after printing to maintain the long-time stability of the three-dimensional structure, thereby preparing the bone flap with the autologous osteoblast three-dimensional scaffold structure.

S4, implanting the bone flap with the autologous osteoblast three-dimensional scaffold structure into the skull defect part of a New Zealand rabbit, continuously proliferating and differentiating the implanted seed cells to generate bone tissues while gradually degrading the biological material, and perfectly fusing the newly generated bone tissues with the defect part to complete defect repair.

4. The method for repairing the skull defect model of the New Zealand rabbit by the autologous osteoblast 3D biological composite printing skull bone flap according to claim 1, wherein the method comprises the following steps: the mass ratio of the pretreated 3D printing powder material to the phosphoric acid solution is 1: 1.2.

5. The method for repairing the skull defect model of the New Zealand rabbit by the autologous osteoblast 3D biological composite printing skull flap according to claim 2, which is characterized in that: the modeling software is 3DMax, the 3D printer adopts an electrostatic spinning technology, the electrostatic spinning technology is a method for electrically jetting and stretching material molecules into filaments, and the electrostatic spinning technology is the only method capable of continuously preparing the nanofibers at present.

Technical Field

The invention belongs to the technical field of skull repair, and particularly relates to a method for repairing a new zealand rabbit skull defect model by using an autologous osteoblast 3D biological composite printing skull flap.

Background

The skull defect refers to a series of clinical symptoms of diseases caused by skull defects due to craniocerebral injuries, skull tumors, other craniocerebral operations and the like, is a common sequela after injuries and operations of neurosurgical patients, exceeds 300 million patients with skull defects caused by traffic accidents and accidents every year, and has the tendency that the expected life of the masses is prolonged along with the continuous increase of economic level and the continuous development of various causes, so that the incidence rate and the detection rate of the craniocerebral injuries, the cerebrovascular diseases and the brain tumors are increased year by year, and the incidence rate of the skull defects is also increased year by year.

(1) When repairing skull defect parts of New Zealand rabbits, the most common skull repairing material at present is a titanium alloy mesh frame, which is widely applied due to strong mechanical property and good histocompatibility, but most of titanium meshes are imported at present, the price is high, the manufacturing process is complex, the manufacturing process firstly utilizes CT scanning data of a patient to establish a skull defect three-dimensional skull model, a resin model is manufactured, manual bending processing is carried out on the titanium alloy mesh frame on the model, the operation is complex, the shaping time is long, the structure is not closed tightly, meanwhile, the repairing effect mainly depends on the clinical experience and the technical level of a doctor, personalized processing and forming cannot be carried out, and the defects that the skull defect is influenced by the fact that a large uncertain structure is difficult to perfectly match with the skull defect of the New Zealand rabbits and the like exist.

(2) The 3D printing technique can only carry out simple bone tissue engineering support and print, do not carry the cell and print simultaneously, non-degradation emulation skull is only as the defective filling material of skull, can't combine together with autologous bone, degradable emulation skull is in internal degradation gradually, absorb and discharge, but the unable defective position of repairing completely of the new born bone of induced generation, be difficult to reach the structure and the mechanical requirement of skull, the malformation healing that forms the bone even bone does not connect etc. it is the urgent big difficult problem that needs to solve to use emulation skull treatment skull defect.

Disclosure of Invention

Technical problem to be solved

In order to overcome the defects of the prior art, the invention provides a method for repairing a new zealand rabbit skull defect model by using autologous osteoblast 3D biological composite printing skull flap, and solves the problems that a 3D printing technology can only perform pure bone tissue engineering scaffold printing, does not carry cells for simultaneous printing, a non-degradable simulation skull only serves as a skull defect filling material and cannot be integrated with autologous bones, a titanium alloy mesh frame is manually bent by adopting titanium alloy mesh frame repairing, the operation is complex, the shaping time is long, the structural alignment is not tight, and meanwhile, the repairing effect mainly depends on the clinical experience and technical level of a doctor and cannot be individually processed and shaped.

(II) technical scheme

In order to achieve the purpose, the invention provides the following technical scheme: the method for repairing the skull defect model of the New Zealand rabbit by the autogenous osteoblast 3D biological composite printing skull flap comprises the following steps:

s1, firstly, taking out autogenous bone from a New Zealand rabbit, separating and culturing osteoblasts as seed cells for later use, and mixing hydroxyapatite powder and polycaprolactone according to a mass ratio of 1.2: 1, and fully mixing to obtain the pretreated 3D printing powder material.

And S2, fully mixing the 3D printing powder material prepared and pretreated in the above step with a phosphoric acid solution to prepare the pretreated 3D printing mixed material.

S3, mixing the prepared pre-processed 3D printing powder material with the pre-processed 3D printing mixed material through a phosphoric acid solution, and storing the mixture in an environment of 18-25 ℃ below zero for standby use to obtain the 3D printing initial material.

As a further scheme of the invention: the 3D model is established by the following steps:

and S1, performing solid scanning on the defected diseased part of the head of the New Zealand rabbit by adopting a 3D scanner to obtain three-dimensional data of the head image of the New Zealand rabbit and recording the three-dimensional data.

And S2, secondly, carrying out 3D modeling by adopting modeling software, importing the three-dimensional data of the head image of the New Zealand rabbit in S1 into the modeling software, and processing the data by the modeling software to obtain a 3D stereo architecture.

As a further scheme of the invention: the 3D printing steps are as follows:

s1, when seed cells are implanted into the 3D printing initial material, the seed cells are printed into a three-dimensional structure, the autologous osteoblasts of the New Zealand rabbit are respectively mixed with agarose, methylcellulose or alginate and a thermal reversible gel material, the cell suspension can be accurately positioned and printed on the surface of the thermal reversible gel through a printer, after the cells are printed on one layer of gel, a new layer of material is covered and solidified, and the process is repeated continuously to obtain a three-dimensional cell structure, namely a cell matrix material mixture.

And S2, introducing the three-dimensional data of the 3D stereoscopic model into a 3D printing device, introducing the stored 3D printing initial material and the cell matrix material mixture into the 3D printing device, and performing printing operation in an environment at minus 25 ℃.

And S3, in the 3D printing process, printing the cell matrix material mixture layer by layer according to a pre-designed model under the control of a computer to form a specific three-dimensional structure, and chemically crosslinking the cell matrix material mixture after printing to maintain the long-time stability of the three-dimensional structure, thereby preparing the bone flap with the autologous osteoblast three-dimensional scaffold structure.

S4, implanting the bone flap with the autologous osteoblast three-dimensional scaffold structure into the skull defect part of a New Zealand rabbit, continuously proliferating and differentiating the implanted seed cells to generate bone tissues while gradually degrading the biological material, and perfectly fusing the newly generated bone tissues with the defect part to complete defect repair.

As a further scheme of the invention: the mass ratio of the pretreated 3D printing powder material to the phosphoric acid solution is 1: 1.2.

As a further scheme of the invention: the modeling software is 3DMax, the 3D printer adopts an electrostatic spinning technology, the electrostatic spinning technology is a method for electrically jetting and stretching material molecules into filaments, and the electrostatic spinning technology is the only method capable of continuously preparing the nanofibers at present.

(III) advantageous effects

Compared with the prior art, the invention has the beneficial effects that: the scheme adopts the 3D biological printing and electrostatic spinning technology of the autogenous osteoblasts, outputs at the same layer and prints in multiple layers to construct a simulated skull flap, so that the printed skull flap is precisely individualized to meet the mechanical requirements of the skull, simultaneously, the cells are uniformly distributed in a three-dimensional space of a bracket to maintain the normal nutrition and metabolism of the cells and ensure the activity of the cells, the printed simulated skull flap is immediately transplanted to repair the skull defect model of the New Zealand rabbit, the histopathology, cellular immunology, imaging and other methods are applied to detect the activity of the osteoblasts, the structural change of the bracket, the fusion condition of the skull flap and the bone margin and the like, and the aim is to explore and repair a brand new biomaterial for the skull defect, establish a new treatment mode for the clinical skull defect repair, and simultaneously detect a 3D biological multi-material, a new method for repairing the skull defect model of the future 3D biological multi-material, The printing of multicellular organism organs provides important experimental basis and theoretical basis.

Detailed Description

The technical solution of the present patent will be described in further detail with reference to the following embodiments.

The invention provides a technical scheme that: the method for repairing the skull defect model of the New Zealand rabbit by the autogenous osteoblast 3D biological composite printing skull flap comprises the following steps:

s1, firstly, taking out autogenous bone from a New Zealand rabbit, separating and culturing osteoblasts as seed cells for later use, and mixing hydroxyapatite powder and polycaprolactone according to a mass ratio of 1.2: 1, and fully mixing to obtain the pretreated 3D printing powder material.

And S2, fully mixing the 3D printing powder material prepared and pretreated in the above step with a phosphoric acid solution to prepare the pretreated 3D printing mixed material.

S3, mixing the prepared pre-processed 3D printing powder material with the pre-processed 3D printing mixed material through a phosphoric acid solution, and storing the mixture in an environment of 18-25 ℃ below zero for standby use to obtain the 3D printing initial material.

The 3D model is established by the following steps:

and S1, performing solid scanning on the defected diseased part of the head of the New Zealand rabbit by adopting a 3D scanner to obtain three-dimensional data of the head image of the New Zealand rabbit and recording the three-dimensional data.

And S2, secondly, carrying out 3D modeling by adopting modeling software, importing the three-dimensional data of the head image of the New Zealand rabbit in S1 into the modeling software, and processing the data by the modeling software to obtain a 3D stereo architecture.

The 3D printing steps are as follows:

s1, when seed cells are implanted into the 3D printing initial material, the seed cells are printed into a three-dimensional structure, the autologous osteoblasts of the New Zealand rabbit are respectively mixed with agarose, methylcellulose or alginate and a thermal reversible gel material, the cell suspension can be accurately positioned and printed on the surface of the thermal reversible gel through a printer, after the cells are printed on one layer of gel, a new layer of material is covered and solidified, and the process is repeated continuously to obtain a three-dimensional cell structure, namely a cell matrix material mixture.

And S2, introducing the three-dimensional data of the 3D stereoscopic model into a 3D printing device, introducing the stored 3D printing initial material and the cell matrix material mixture into the 3D printing device, and performing printing operation in an environment at minus 25 ℃.

And S3, in the 3D printing process, printing the cell matrix material mixture layer by layer according to a pre-designed model under the control of a computer to form a specific three-dimensional structure, and chemically crosslinking the cell matrix material mixture after printing to maintain the long-time stability of the three-dimensional structure, thereby preparing the bone flap with the autologous osteoblast three-dimensional scaffold structure.

S4, implanting the bone flap with the autologous osteoblast three-dimensional scaffold structure into the skull defect part of a New Zealand rabbit, continuously proliferating and differentiating the implanted seed cells to generate bone tissues while gradually degrading the biological material, and perfectly fusing the newly generated bone tissues with the defect part to complete defect repair.

The mass ratio of the pretreated 3D printing powder material to the phosphoric acid solution is 1: 1.2.

The modeling software is 3DMax, the 3D printer adopts an electrostatic spinning technology, the electrostatic spinning technology is a method for electrically jetting and stretching material molecules into filaments, and the electrostatic spinning technology is the only method capable of continuously preparing the nanofibers at present.

Although the preferred embodiments of the present patent have been described in detail, the present patent is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present patent within the knowledge of those skilled in the art.