CN210932936U

CN210932936U - Tissue engineering bone

Info

Publication number: CN210932936U
Application number: CN201920467199.7U
Authority: CN
Inventors: 宋滇文; 张传忠; 车凌宾; 周磊; 刘红梅
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-03-29
Filing date: 2019-03-29
Publication date: 2020-07-07
Anticipated expiration: 2029-03-29

Abstract

The utility model relates to a tissue engineering bone, including support material, seed cell and the biological factor that promotes the growth of seed cell, set up in support material spatial structure and be connected with nutrition conveyor can give the seed cell in the tissue engineering bone and by the bone histiocyte of seed cell growth go out effectively supply with nutrient substance's nutrient substance pipeline. The space structure of the bracket material with the nutrient substance conveying pipeline is manufactured by manufacturing the shape of the tissue engineering bone according to the shape required by the transplanted bone tissue by using the forming technology such as 3D printing or mechanical carving. After the tissue engineering bone is implanted into the bone defect part, the connection with the nutrition conveying device is continuously kept, and the in-vitro nutrition conveying is continuously carried out. Compared with all the existing tissue engineering bone technologies, the disease course can be obviously shortened. This technique is equivalent in a sense to the fabrication of pedicle bone tissue flaps by tissue engineering.

Description

Tissue engineering bone

Technical Field

The utility model relates to a medical tissue engineering composite material.

Background

Bone defects due to trauma and surgery are common clinical problems. Severe bone defects can lead to delayed bone healing and nonunion and other complications, with high disability rate. At present, for the treatment of bone defects, autologous bone grafting, allogeneic bone grafting and other methods are commonly used for filling and repairing, but the autologous bone grafting has fewer sources and limited bone taking quantity; allogenic bone transplantation has immunogenicity, and is easy to cause rejection reaction of organisms. The artificial bone substitute such as hydroxyapatite, calcium phosphate and the like has been successfully applied to clinic, but the main effect of the artificial bone substitute is filling, supporting or bone conduction, and the artificial bone substitute has the defects of weak bone induction capability, poor physiological repair capability and the like. Therefore, the development of new biomaterials with strong bone induction capability and repair capability becomes a new challenge in the field of orthopedics and repair and reconstruction surgery at present.

The tissue engineering bone is a cell hybrid material formed by planting seed cells on a cell scaffold or extracellular matrix which has good biocompatibility and can be gradually degraded and absorbed by a human body after in vitro culture and amplification. The cell hybrid material is implanted into the bone defect part, and the implanted bone cells are continuously proliferated while the cell scaffold material is gradually degraded, so that the aim of repairing the bone tissue defect is fulfilled.

In the current tissue engineering bone technology, osteoblasts, bone marrow mesenchymal stem cells (BMSCs), Adipose Derived Stem Cells (ADSCs), embryonic stem cells (ES), and the like are generally used as bone tissue engineering seed cells; the scaffold material is prepared by processing natural biological tissues by a series of physical and chemical methods by using artificial synthetic materials such as calcium-phosphorus ceramics, polylactic acid and the like or natural biological derived materials, such as natural bones, coral bones and the like, and is disclosed in patent document CN 101032430A; a composite material added with biological factors or biological factors such as Bone Morphogenetic Protein (BMP), Vascular Endothelial Growth Factor (VEGF), and a composite material introduced with genetic engineering technology, and finally forms bone tissue by in vivo construction, in vitro construction, or in situ tissue construction, see patent document CN103768656A, non-patent document "background and progress of bone research in tissue engineering" journal of injury and repair (electronic edition) of china, volume 8, No. 2013, No. 5. However, the tissue engineered bone cultured by this method is disordered and has no thin layer of original bone tissue of proper tissue structure and mechanical structure. However, the lack of blood supply system in the bone tissue constructed in vitro is a key factor influencing the success of bone tissue construction and the survival of the constructed bone tissue. When the volume of the constructed tissue is too large, ischemia and hypoxia are easy to occur to cause death of seed cells, and the construction and formation of bone tissues are influenced finally.

At present, vascularization of tissue engineering bone is a key link for successful construction of bone tissue engineering. The tissue valve which is easy to be used in the body of a receptor, such as a pedicel fascia valve, is cultured into the pedicel bone tissue valve through artificial bone engineering for autografting, which is a relatively mature method for solving the problem at present, and is published in China clinical rehabilitation Vol.9, No. 30, 2005-08-14, of patent document CN107988151A, non-patent document research on revascularization of tissue engineering bone. However, this approach requires surgery to destroy the normal tissue of the recipient, increases the patient's pain and the various risks associated with the surgery, and seems to be prohibitively expensive compared to the starting point of tissue engineering bone techniques to promote healing of bone tissue defects with reduced surgical workload and patient pain. The bone tissue construction technique in this way is excluded from the definition of the tissue engineering bone technique in the present case.

Disclosure of Invention

The utility model discloses a tissue engineering bone. The tissue engineering bone has similar shape and size with the defected bone tissue, and includes rack material, seed cell and required biological factor, and conveying pipeline capable of being connected to the nutrient conveying device and capable of supplying nutrient to the seed cell in the tissue engineering bone and the bone tissue cell grown from the seed cell.

The preparation method of the tissue engineering bone utilizes a forming technology to prepare the shape of the tissue engineering bone containing the bracket material, the seed cells and the necessary biological factors according to the shape required by the transplanted bone tissue, and simultaneously constructs a conveying pipeline which can be connected with a nutrition conveying device and can effectively supply nutrient substances to the seed cells in the tissue engineering bone and the bone tissue cells grown by the seed cells in the forming process.

The forming technology can use common 3D printing technology or mechanical carving forming.

The scaffold material of the tissue engineering bone can adopt any existing tissue engineering bone scaffold material which is proved by experiments and has the necessary physicochemical and biological characteristics of mechanical strength, pore characteristics, tissue compatibility, in-vivo degradability and the like.

The seed cells in the tissue engineering bone can be poured and attached after the scaffold material is formed, and can also be directly planted in the gap of the scaffold during 3D printing.

The nutrient substance conveying pipeline in the tissue engineering bone can simulate the size and the distribution rule of a blood vessel system of natural bone tissue, and can also be manufactured into any form and distribution rule form by only taking effective nutrient substance conveying as a principle.

The nutrient delivery line need not have a complete structure resembling the arterial-capillary-venous system, but need only have a form resembling one of the arterial-capillary, or capillary-venous systems. One end of the main pipeline is communicated with the nutrition conveying device.

The nutrient channel is connected with the nutrient input device, nutrient solution uniformly reaches each part of the tissue engineering bone through a system similar to artery-capillary, and the exhausted nutrient solution after completing the material exchange seeps out through the basic pore or seeps into the basic pore and is sucked out by the negative pressure device through a nutrient pipeline similar to capillary-vein system.

The skeleton pores in the tissue engineering bone for accommodating the planted cells and the bone tissue cells bred by the planted cells are called as basic pores, and when the tissue engineering bone is formed by a 3D printing technology, the set specific pore size and porosity can refer to the pore size and porosity of normal cancellous bone tissues of an experimental animal or human body.

In general, the seed cells are usually obtained from autologous tissues of experimental animals or surgical patients, and can be obtained from allogeneic sources under the condition that the rejection reaction is not problematic and/or the conditions for obtaining the autologous tissues are not available.

After being prepared in the tissue engineering bone, the tissue engineering bone is generally cultured in vitro, the growth state of cells is stabilized, the differentiation and growth conditions of the cells are observed, the conditions that the tissue engineering bone cannot be implanted into a recipient body without pollution, necrosis and the like are determined, and then the tissue engineering bone is implanted into a bone defect part under the condition that the tissue engineering bone is kept connected with a nutrition conveying device and is continuously supplied with in vitro nutrition conveying.

After the tissue engineering bone technology is completely mature, the clinically needed engineering bone graft is printed and can reach proper mechanical strength, and the tissue engineering bone graft can be directly implanted into a receptor without an in-vitro culture observation process.

The in vitro nutrition delivery is continued until the recipient's own vascular tissue grows into each part of the tissue engineering bone and is enough to provide necessary nutrition for the tissue engineering bone, and gradually reduced until the in vitro nutrition input is stopped, and an in vitro nutrition delivery pipeline connected with the tissue engineering bone is removed.

When the amount of tissue engineered bone mass required for surgery is large, a nutrient channel like an artery-capillary, or capillary-vein system for the infusion of nutrients can be provided in addition to the basal pores of normal bone tissue during 3D printing. When the tissue engineering bone is implanted into a receptor, the nutrient supply and the metabolite discharge through the nutrient channel during in vitro culture are still maintained. The nutrition system continues to work until the recipient's own vascular tissue grows into the tissue-engineered bone and gradually grows to a point where it is functionally sufficient to replace the nutrient supply from outside the body.

The technology has the positive effects that a tissue engineering bone with a larger size is cultured, the tissue engineering bone is implanted into a body under the state of keeping the whole biological activity of the tissue engineering bone, the natural transition is carried out from the in vitro nutrition delivery to the nutrition supply and the nutrition supply by a receptor, the healing process of bone defect is mainly the process of reconstructing the whole tissue engineering bone to a normal bone tissue, and the process of creeping and extending the bone tissue along a tissue engineering bone scaffold in space is spanned. Compared with all the existing tissue engineering bone technologies, the disease course can be obviously shortened. This technique is equivalent in a sense to the fabrication of pedicle bone tissue flaps by tissue engineering.

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

Drawings

FIGS. 1-2 are schematic diagrams of Mesenchymal Stem Cells (MSCs) as seed cells, controlling the mechanical strength of a cell matrix, alginate hydrogel, by adjusting the concentration of calcium ions and the concentration of phosphate functional groups to control the osteogenic differentiation of the MSCs, and finally forming tissue-engineered bones having a potent strength.

Fig. 3 is a schematic diagram showing the basic structure of a tissue engineering bone formed by 3D printing, a tree-shaped nutrient delivery pipeline which is printed according to the blood vessel distribution rule of normal bone tissues and communicated with a preset nutrient delivery pipeline, and an artery-capillary vessel mode.

Fig. 4 is a schematic diagram, similar to the structure of fig. 3, of a capillary-vein pattern.

Fig. 5 is a schematic view showing the basic structure of a tissue engineering bone formed by mechanical carving, and only one nutrition delivery pipeline is positioned at the central part vertically and a base gap arranged in a radial mode, namely an artery-capillary vessel mode.

Fig. 6 is a schematic diagram, similar to the structure of fig. 3, of a capillary-vein pattern.

Fig. 7 is a schematic view showing the tissue-engineered bone of fig. 5 implanted in a human body in an artery-capillary mode.

Fig. 8 is a schematic view, similar to fig. 7, of a capillary-vein pattern.

Fig. 9 is a schematic view, and fig. 7 is a partial enlargement.

Detailed Description

Example 1

In the example, Mesenchymal Stem Cells (MSCs) are used as the seed cells of the tissue engineering bone, and the osteogenic differentiation of MSCs is controlled by controlling the mechanical strength (expressed by elastic modulus) of the cell matrix, alginate hydrogel, and the concentration of phosphate functional groups, and finally, a large-sized tissue engineering bone having sufficient strength for implantation into a bone defect site is formed.

Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, belong to pluripotent stem cells, and can differentiate into various tissues such as fat, bone, cartilage and the like under specific induction conditions in vivo or in vitro. The stem cell has the advantages of convenient material acquisition, small damage to donors, easy separation and culture, strong in vitro proliferation capacity, multidirectional differentiation potential after continuous subculture and cryopreservation and the like, and becomes the stem cell with the greatest clinical application prospect in the fields of cell biotherapy, tissue regeneration engineering and the like. Extracellular matrix (ECM) is a macromolecule synthesized and secreted by cells, distributed on the cell surface or between cells, and has a major component of polysaccharide, protein, or proteoglycan. These materials form a complex network structure and play a decisive role in the differentiation and growth of cells. Therefore, the material bracket can be used for simulating the surrounding of the extracellular matrix around the MSCs to construct a novel composite tissue engineering bone. Such studies are currently focused mainly on promoting osteogenic differentiation of MSCs by cytokines, proteins (e.g., Bone morphogenic protein, BMP). One method is to directly add exogenous proteins such as BMP into the scaffold material to regulate the osteogenic differentiation of MSCs by slow release. However, the method has the disadvantages of high protein cost, short effective period, difficult effective controlled release of the protein and the like. In another method, the gene for promoting bone action is introduced into the MSCs by gene transfection method, so that the target gene is expressed in the MSCs and synthesizes the growth factor with bone induction action, thereby overcoming the defects of short half-life period in vivo, repeated administration and the like of exogenous growth factor. However, transfection with viral vectors has a safety risk despite high transfection efficiency; the non-viral vector has the defects of low transfection efficiency, poor stability and the like. The differentiation of mesenchymal stem cells is influenced by various factors, and recent research shows that besides the growth factors and proteins, the differentiation of MSCs is also regulated and controlled by physical and chemical signals such as mechanical strength and chemical groups of the scaffold material. The existing research results show that the osteogenic differentiation of the MSCs can be controlled by controlling the mechanical strength (expressed by elastic modulus) and the concentration of phosphate functional group-containing groups of the cell matrix or culture medium such as alginate hydrogel, and finally the tissue engineering bone is formed.

See figures 1-2. The specific method is that a 3D printing technology is utilized, a hydrogel material which can control the elastic modulus after solidification and MSCs are made into a tissue engineering bone culture body with a shape required clinically, the MSCs in the culture body are observed and determined to survive under artificial culture conditions and begin to differentiate directionally to osteoblasts, and then the tissue engineering bone is used clinically.

In the printing process, the cell mixed liquid carrying the MSCs and the liquid gel precursor forming the gel are conveyed to a printing head through different material channels of a printer, a tissue engineering bone matrix framework with proper gap size and porosity is formed through preset 3D model data, and the MSCs are distributed in the pores of the matrix framework at proper density. The interstitial skeleton pores are called basic pores, and the specific pore size and porosity are referred to the pore size and porosity of the normal cancellous bone tissues of the experimental animals or human bodies.

See fig. 3-4. While the tissue engineering bone basic structure 11 containing the mesenchymal skeleton and the MSCs cells is formed by 3D printing, a tree-shaped nutrient delivery pipeline 12 communicated with a preset nutrient solution delivery pipeline is printed out approximately according to the blood vessel distribution rule of normal bone tissues. Under the drive of the peristaltic pump 15, the nutrient solution 14 can flow in the direction of the delivery pipe 13, the tree-shaped nutrient delivery pipeline main pipe, the tree-shaped nutrient delivery pipeline branches, the interstitial skeleton pores and the outside of the tissue engineering skeleton body, as shown in fig. 3. Flow in the opposite direction is also possible, as in fig. 4. In the figure, 16 is a tissue engineering bone incubation container, and 17 is a tissue engineering bone incubation container cover.

Example 2

The tissue engineering bone prepared by the method described in example 1 requires a tissue 3D printer with extremely high resolution and harsh temperature control and aseptic conditions, and the tissue engineering bone formed by printing usually has low mechanical strength, and the mechanical strength of the tissue engineering bone needs to be enhanced by the bone tissue generated by tissue culture, which means long time in vitro culture.

See fig. 5-6. The appropriate tissue scaffold 21 may be fabricated first according to the shape of the bone defect and then the seed cells injected or infused into the scaffold. The spatial structure in the scaffold also need not simulate the gap structure of natural bone tissue, as long as the basal gap 23 of appropriate porosity and pore size and nutrient delivery conduit of reasonable size and distribution can be formed. In the figure there is only one nutrient transport line 22 located in the central part, longitudinally above and below, and radially arranged base gaps 23. The nutrient solution 24 was delivered in the same manner as in example 1.

The tissue scaffold can be made by carving natural bone floss material without organic matter, or synthetic polymer material with good tissue compatibility such as polylactic acid, or natural biological material such as alginate hydrogel. More than two materials can be adopted for printing, and the affinity and the higher mechanical strength of the seed cells are considered.

When the tissue engineering bone prepared by the method has enough strength, after the seed cells are inoculated, the operation of implanting the receptor can be implemented only by observing that the cells survive and are successfully distributed in the whole tissue engineering bone.

Tissue engineered bone 31 is conventionally anchored to the bone defect site by internal fixation instrument 38 during surgery, through a tunnel cut through normal bone tissue 39 and through exit nutrient delivery tube 331, see fig. 7-9.

In the early stage after the implantation operation, nutrient substances are positively pressurized and conveyed to the nutrient conveying pipeline 32 in the center of the tissue engineering bone through the peristaltic pump 35 and the nutrient conveying pipeline 331, and the wound drainage tube 332 negatively pressurized and drained nutrient-poor liquid and tissue exudate to the negative pressure suction device 333. In the later period after operation, the wound exudate flowing out of the drainage tube is changed from transparent liquid converted from blood into nutrient solution which is infused through the wound drainage tube and is sucked and discharged through the nutrient delivery tube of the tissue engineering bone under negative pressure. Therefore, the tissue exudate of the wound can be used as nutrient solution, and the new blood vessels of the wound tissue can be favorable for rapidly growing into the tissue engineering bone.

By shooting or CT examination, the nutrient solution infusion is gradually reduced when the peripheral part of the tissue engineering bone begins to form new bone tissue, and the nutrient solution infusion can be stopped when the central part in the tissue engineering bone also generates remarkable new bone tissue, and the nutrient delivery pipe is pulled out.

Claims

1. A tissue engineering bone comprises a bracket material, seed cells and biological factors for promoting the growth of the seed cells, and is characterized in that a nutrient substance conveying pipeline which is connected with a nutrient conveying device and can effectively supply nutrient substances to the seed cells in the tissue engineering bone and the bone tissue cells grown from the seed cells is arranged in a space structure of the bracket material.

2. The tissue-engineered bone according to claim 1, wherein the nutrient delivery channel is a tree-like channel like one of the arterial-capillary or capillary-venous systems, the main end of the channel being in communication with a nutrient delivery device.

3. The tissue engineering bone according to claim 1, wherein the nutrient delivery line is composed of a nutrient delivery line trunk located at a central portion and a radially arranged basal gap, the nutrient delivery line trunk being in communication with a nutrient delivery device.

4. The tissue engineering bone according to claim 1, wherein the nutrient delivery line is formed by a molding technique to conform to the shape of the bone tissue to be transplanted and is constructed during the molding process.

5. The tissue-engineered bone according to claim 4, wherein the molding technique is a 3D printing technique or a mechanical engraving molding technique.

6. The tissue-engineered bone of claim 5, wherein the seed cells are perfused and attached in the interstices of the scaffold after the scaffold material is formed.

7. The tissue engineered bone of claim 5, wherein said seed cells are "seeded" directly into the scaffold gap at the time of 3D printing.