AU2015101218A4

AU2015101218A4 - A cell-scaffold complex and 3D printing fabrication method

Info

Publication number: AU2015101218A4
Application number: AU2015101218A
Authority: AU
Inventors: Hailong Hu; Minchao Shi; Hong Wang; Nan Zhang
Original assignee: QINGDAO UNIQUE PRODUCTS DEVELOP CO Ltd
Current assignee: QINGDAO UNIQUE PRODUCTS DEVELOP CO Ltd
Priority date: 2015-06-11
Filing date: 2015-09-03
Publication date: 2015-10-08
Anticipated expiration: 2023-09-03
Also published as: CN104888277B; CN104888277A

Abstract

Abstract A cell-scaffold complex and 3D printing fabrication method comprises a biological scaffold resemble with actual defects of autologous organs or tissues, nanofibrous layers printed on the biological scaffold capable of mimic natural extracellular matrix, which are beneficial to cells for adhering to, growing and proliferating and cell suspension printed on the nanofibrous layers. The required cell-scaffold complex can be accurately printed in accordance to the actual defects of patients' organs or tissues through the 3D printing fabrication method.

Description

- 1 A Cell-Scaffold Complex and 3D Printing Fabrication Method Field of the invention [0001] The present invention belongs to the technical field of biotechnology, and particularly relates to a cell-scaffold complex and a 3D printing fabrication method thereof. Background art [0002] Medical breakthroughs in the 2 0 th Century are represented by repairing of cell defects and organ transplantation. Approximately one million of suffers living in the shadow of death are given a new life by receiving an organ from another body. But the number of those who died due to lack of donor organs are up to tens of millions, no to mention hundreds of millions patients who are eager to be treated by cell repairing. Hence, someone believes that the 2 1 st century will be a century of tissue repairing and organ transplantation. However, the development of 3D printing technology gives those hundreds of millions people who need help from tissue repairing and organ transplantation a glimpse of hope. [0003] The shapes of scaffold have been produced in a controllable manner which could satisfy the requirements for preparing gradient scaffolds based on sufficient technology principles. But the sizes of pores produced via such manner are comparatively large, and in particular pores with diameters lower than 100pm could not be prepared for. Therefore micro environment for cell-growth could not be provided with, and cells are difficult to be adhered to. Normally, homogenous material and man-made complex material prepared beforehand are used in 3D printing which are relatively limited in forming scaffolds with material gradient characteristics. Hence, the development of bone scaffold forming techniques on the basis of electro spinning is the fastest section in the field of biotechnology. Electro spinning uses the effect of strong electrical field to draw steams of liquid from polymer solution or melt. Electro spinning is a novel processing method in producing fiber on nanometer scale. The applications of electro spinning have related to the fields of bioscience, tissue engineering, photoelectric devices, aircraft engineering and the like. Fibers on the scales of sub-micron, or even nanometer could be drawn by electro spinning so as to form a structure of 3D micro pores communicated with each other, to which is suitable for cells grow or adhere. However, the shape of scaffold formed from fibers produced by the jet from electro spinning is resemble with chaotic nonwovens, and thereby being difficult to be controlled in a subtle way, In conclusion, obviously any one of the traditional processing methods cannot realize the formation on -2 different sizes, therefore the requirements for forming macro shape of bone scaffold and micro pores could not been satisfied simultaneously. Summary of the invention [0004] The object of the present invention is to overcome the shortcomings described above. The present invention provides a cell-scaffold complex and a 3D printing fabrication method thereof, which combining 3D printing with electro spinning. The present invention will further boost the scope of applications of 3D biology printing technology in tissue engineering and give patients waiting to be treated with tissue recovery and organ transplantation a glimpse of hope of healing and a new life. [0005] The present invention provides a cell-scaffold complex. The cell-scaffold complex comprises a biological scaffold, wherein nanofibrous layers are provided onto the biological scaffold and biology cell suspension is provided onto the nanofibrous layers. [0006] Further, the porosity of the biological scaffold is in the range of 50% to 90%, the pore diameter of the biological scaffold is in the range of 50 to 500pm, and the pore diameter of the nanofibrous layer is in the range of 0.1 to 10pm. [0007] Further, the biological scaffold consists of one or more of biopolymer material, natural biomaterial and inorganic material, wherein the biopolymer material refers to at least one of poly L-lactic acid (PLLA), poly lactic-co-glycolic acid (PLGA) or polycaprolactone (PCL); the natural biomaterial refers to at least one of gelatin, chitosan or sodium alginate; the inorganic material refers to at least one of tertiary calcium phosphate (TCP) or Nano hydroxyapatite (HA). [0008] Further, the nanofibrous layer consists of one or a mixture of chitosan, collagen and polyvinyl alcohol. [0009] Further, the mass ratio between collagen and chitosan is 1 to 5:1. [0010] Further, the mass ratio between polyvinyl alcohol and chitosan is 1 to 5:1. [0011] Further, the cell suspension comprises cell culture medium, serum and autologous cells; the mass ratio between cell culture medium and serum is in the range of 2 to 6:1, and the -3 cell concentration is in the range of 10 4 /ml to 10 6 /ml; wherein the cells are osteoblast, corneal stromal cells or adipose-derived stem cells. [0012] The present invention also provides a 3D printing fabrication method of a cell scaffold complex, comprising the following steps: [0013] Step 1, forming a cell biological scaffold: blending raw materials of the cell biological scaffold evenly; producing a porous biological scaffold resemble with actual defects of tissues or organs by processing the blended raw materials layer by layer with scaffold printing head of bio-printer; [0014] Step 2, forming nanofibrous layers: dissolving nanofibrous layer material into deionized water at a certain ratio to produce electro spinning solution; spraying the electro spinning solution onto each layer or into gaps between layers by electrostatic spinning nozzle to form nanofibrous layers on the biological scaffold in a layer-by-layer manner; [0015] Step 3, cell printing: spraying the cell suspension on the nanofibrous layer by cell printing head and forming the cell-scaffold complex by adding up layers. [0016] Further, adjacent pores inside the porous biological scaffold are connected communicated with each other. [0017] Further, the mass concentration of the electro spinning solution is in the range of 6% to 10%, and wherein the percentage of chitosan in the electro spinning solution is in the range of 0% to 5%, and the percentage of collagen in the electro spinning solution is in the range of 0% to 8% and the percentage of polyvinyl alcohol in the electro spinning solution is in the range of 0% to 10%. [0018] Compared with the prior art, the advantages and beneficial effects of the present invention are illustrated as follows. Cells of the cell-scaffold complex are collected from patients themselves. The cells are added into cell culture solution including serum and printed by piezoelectric techniques onto surfaces of nanofibrous layer on a biological scaffold which is printed by another printing head. Required cell-scaffold complex for patients can be accurately printed in accordance with actual defects of patients' organs or tissues by utilizing the printing fabrication method. The loading cells are collected from patients own that can effectively avoid the problem of immunological rejection. Further, nanofibrous layers which can mimic the -4 environment of extracellular matrix are provided in cell printing, and the number and position of cells inside the biological scaffold are controllable. [0019] The cell-scaffold complex 3D printing fabrication method comprises the following steps: firstly, printing a biological scaffold to fulfill accurate control of the scaffold shape; then producing nanofibrous layers with good compatibility on each layer or every several layers of the biological scaffold by electro spinning technology; and then a cell-scaffold complex can be fabricated, wherein the number or internal position of cells in the biological scaffold can be controlled, and nanofibrous layers structures capable of effectively mimicking extracellular matrix can be produced inside by quantitatively spraying cell suspension on the nanofibrous layers. The shape or the outline of the structures of nanofibrous layers and the macroporosity could be accurately controlled, and further the structures of nanofibrous layers have good mechanical properties. The difficulty of cell growth and adherence can be efficiently eased and the problem of cell suspension can be solved through the method described above. Brief Description of the Drawings [0020] Various embodiments of the invention will be described with reference to the following drawings, in which: Figure 1 is a SEM observation photo (x20) of PCL/HA complex scaffold; Figure 2 is a SEM observation photo (x50) of PCL/HA complex scaffold; Figure 3 is a SEM observation photo (x500) of the collagen-chitosan electro spinning nanofibrous layer; Figure 4 is a SEM observation photo (x2000) of the collagen-chitosan electro spinning nanofibrous layer. Detailed Description of the Embodiment [0021] A detailed description of the invention will be disclosed as follows with reference to specific embodiments. [0022] The invention provides a cell-scaffold complex and a 3D printing fabrication method thereof. Such sections with organ defects could be replaced by the cell-scaffold complex to accelerate the growth and proliferation of cells in the defective sections and those printed onto the biological scaffold.

-5 [0023] The cell-scaffold complex in present invention includes a biological scaffold. The biological scaffold is printed and formed by a bio-printer in according with the actual defects in patients' organ. Nanofibrous layers are provided onto the surface of the biological scaffold, wherein the nanofibrous layers are processed and formed by electro spinning. The nanofibrous layer may mimic natural extracellular matrix for facilitating the growth and proliferation of cells adhering to. Biology cell suspension including growth factor is printed onto the nanofibrous layer. The porosity of the biological scaffold ranges from 50% to 90%, and its pore diameter ranges from 50 to 500pm. The pore diameter of the nanofibrous layer on the biological scaffold drawn by electrostatic spinning nozzle ranges from 0.1 to 10pm. [0024] The biological scaffold is composed of one or more of biopolymer materials, natural biomaterials and inorganic materials, wherein the biopolymer material refers to at least one of poly L-lactic acid (PLLA), poly lactic-co-glycolic acid (PLGA) or polycaprolactone (PCL); the natural biomaterial refers to at least one of gelatin, chitosan or sodium alginate; the inorganic material refers to at least one of tertiary calcium phosphate (TCP) or Nano-hydroxyapatite (HA). [0025] The biological scaffold is preferably produced from PCL-nHA with a mass ratio in the range of 2 to 5:1, preferably 4:1. Haydroxyapatite (HA) is an important inorganic constituent of human or animal skeleton and teeth, which has a good performance in bioactivity, compatibility and osteoconduction. But HA can not satisfy the requirements of osseous tissue scaffold material due to its high brittleness, low intensity of load and poor mechanical property. PCL is a type of aliphatic polyester with beneficial properties as good biosafety and mechanical property which is biodegradable and easy to be digested and metabolized by living beings. PCL-nHA composite material is suitable for osseous tissue scaffold material. [0026] The biological scaffold is preferably produced from PLLA-TCP with a mass ratio in the range of 3 to 6:1, preferably 5:1. TCP has high compatibility, but with a shortcoming that its speed of degradability in vivo is overly fast. PLLA is a polymer with low degradation, good strength of extension, high molecular weight and modulus (approximately 4.8 GPa) and strong hydrophobicity. PLLA-TCP composite material can be expected to utilize as cartilage material in tissue engineering. [0027] The nanofibrous layer is preferably produced by collagen-chitosan with a mass ratio in the range of 1 to 5:1, preferably 3:1. The material of nanofibrous is soluble in water to prepare electro-spinning solution with a mass concentration of 10%. Collagen is the richest -6 type of protein inside animals, and also serves as an important constituent of extracellular matrix with advantages of good bionic characteristics, low immunogenicity, good histocompatibility and tissue affinity, and further with a better performance in promoting cell proliferation. The chitosan is a natural biomaterial with good compatibility, a capability to guide bone cells to creep and crawl, and also good hydrophilia, thereby capable of accelerating wound healing and absorption effect. However, the chitosan is poor in mechanical property and solubility property, further difficult for cells to adhere to. Those disadvantages limit applications of chitosan. But the composite electron-spinning fiber layer could mimic the environment of extracellular matrix to promote the adherence and proliferation of cells. [0028] The nanofibrous layer is preferably produced by polyvinyl alcohol-chitosan with a mass ratio in the range of 1 to 5:1, preferably 4:1. The material of nanofibrous is soluble in water to prepare electro-spinning solution with a mass concentration of 8%. Chitosan is good in antimicrobial activity and also could promote wound healing. But chitosan is difficult to be prepared independently, and therefore non-toxic degradable high polymer material PVA with good fiber formality and compatibility can be used together with chitosan to produce a biological membrane scaffold. [0029] The cell suspension comprises cell culture medium, serum and the patients' own cells. The mass ratio between cell culture medium and serum is in the range of 2 to 6:1, and the cell concentration is in the range of 10 4 /ml to 10 6 /ml; wherein the cells are osteoblast, corneal stromal cells or adipose-derived stem cells. [0030] A 3D printing fabrication method for printing the cell-scaffold complex comprises the following steps: [0031] Step 1, forming the cell biological scaffold: blending raw materials of the cell biological scaffold evenly; producing a porous biological scaffold resemble with actual defects of tissues or organs by processing the blended raw materials layer by layer with scaffold printing heads of bio-printer. Adjacent pores are connected with each other in the process of 3D printing. The diameter of the bio-printer head is in the range of 100 to 500pm, and diameters of the forward pore and lateral pore are both in the range of 50X50pm to 500 X 500pm, preferably the diameter of the forward pore is 200 X 200pm and that of the lateral pore is 200X 100pm.

-7 [0032] Step 2, forming the nanofibrous layer: dissolving nanofibrous layer material into deionized water at a certain ratio to produce electro spinning solution; spraying the electro spinning solution onto each layer or into gaps between layers by electrostatic spinning nozzle to form nanofibrous layers, the pore diameter is in the range of 0.1 to 1pm, and the nanofibrous layer on the biological scaffold is formed in a layer-by-layer manner; the mass concentration of the electro spinning solution is in the range of 6% to 10%, and wherein the percentage of chitosan in the electro spinning solution is in the range of 0% to 5%, and the percentage of collagen in the electro spinning solution is in the range of 0% to 8% and the percentage of polyvinyl alcohol in the electro spinning solution is in the range of 0% to 10%. [0033] Step 3, cell printing: spraying the cell suspension on the nanofibrous layer by piezoelectric cell printing head and forming the cell-scaffold complex layer on layer. Due to the fact that, normally, diameters of human cells are in the range of 20 to 30pm. A cell printing head with overly small diameter could be easily plugged, while it also could lead to inaccurate position of cell printing with an overly large diameter. Hence, the diameter of cell printing head in present invention is in the range of 100 to 150pm. [0034] The detailed preparation method for producing the cell suspension comprise the following steps: transferring culture flask with cells inside a clean bench; removing deposit culture solution; adding into trypsin and digesting for one minute, and then removing the trypsin; adding into DMEM/F12 culture solution with 20% serum to terminate digestion; pipetting cells down softly and pipetting the cell suspension repeatedly for three to five times so as to re-suspend the cells. [0035] Embodiment 1 [0036] 1. preparing PCL-nHA composite materials, wherein the mass ratio of PCL and nHA is 4:1: weighing 4g PCL and adding the PCL into 36g dichloromethane solution; stirring the blended with magnetic stirring apparatus until intensive dissolving; adding into 1g nHA and stirring the blended with magnetic stirring apparatus until dissolution uniformity; vacuum deforming and putting into ventilating cabinet to allow organic solvent to volatilize fully; leaving it standby. [0037] 2. preparing collagen-chitosan electro-spinning solution: dissolving collagen-chitosan into deionized water with a mass ratio 1:2 to produce electro-spinning solution with a mass fraction of 8%.

-8 [0038] 3. preparing osteocyte suspension: transferring culture flask with osteocyte inside a clean bench; removing deposit culture solution; adding into trypsin and digesting for one minute and removing the trypsin; adding into DMEM/F12 culture solution with 20% serum to terminate digestion, namely the mass ratio between the culture solution and serum is 4:1 with a cell concentration in the range of 10 4 /ml to 10 6 /ml; pipetting cells down softly and repeatedly and pipetting the cell suspension for three to five times so as to re-suspend the cells. [0039] 4. preparing a osteocyte biological scaffold complex by 3D printing: putting PCL/HA composite scaffold materials into the scaffold printing cylinder of bio-printer; putting collagen chitosan electro-spinning solution for the nanofibrous layer into the electrostatic spinning nozzle; putting osteocyte suspension into piezoelectric cell printing head. The diameter of the biological scaffold head is 300pm, the cell printing head is 120pm, the diameter of forward pore is 300pmX300pm (as seen in the FIG 1 and FIG 2) and the diameter of lateral pore is 300pmX100pm. Under the control of GCODE files (a programming language for controlling the 3D printer), a layer of lattice produced from PCL/HA composite scaffold material is formed on the printing platform by the scaffold printing head of a bio-printer, and then electro spinning material is sprayed by the electro spinning nozzle onto the PCL/HA composite scaffold to form the nanofibrous layer (as seen in the FIG 3 and FIG 4); and then osteoblast is sprayed on the nanofibrous layer by spraying the osteoblast suspension from the cell printing head. [0040] The porosity of the printed and formed PCL/HA composite scaffold measured by mercury injection apparatus is 83.4%. As seen in FIG 2, the pore diameter of the PCL/HA composite scaffold printed and formed is 300pm. As seen in FIG 4, the pore diameter of the collagen-chitosan electro spinning nanofibrous layer is in the range of 1 to 10pm, which is less than the cell diameter. The structure and function of collagen-chitosan electro spinning nanofibrous layer is resemble with those of extracellular matrix. The nanofibrous layer provides an ideal model for cells to adhere to, proliferation and grow. The above process is repeated to form a cell-scaffold complex by adding up layers. [0041] Although a preferred embodiment of the invention has been specifically illustrated and described herein, it is to be understood that minor variations may be made in the invention without departing from the spirit and scope of the invention, as defined the appended claims.

Claims

1. A cell-scaffold complex, characterized in that comprises a biological scaffold, wherein nanofibrous layers are provided onto the biological scaffold and biology cell suspension is provided onto the nanofibrous layers.

2. A cell-scaffold complex according to the claim 1, characterized in that the porosity of the biological scaffold is in the range of 50% to 90%, the pore diameter of the biological scaffold is in the range of 50 to 500pm, and the pore diameter of the nanofibrous layer is in the range of 0.1 to 10pm.

3. A cell-scaffold complex according to the claim 1, characterized in that the biological scaffold is produced from one or more of biopolymer material, natural biomaterial and inorganic material, wherein the biopolymer material refers to at least one of poly L-lactic acid (PLLA), poly lactic-co-glycolic acid (PLGA) or polycaprolactone (PCL); the natural biomaterial refers to at least one of gelatin, chitosan or sodium alginate; the inorganic material refers to at least one of tertiary calcium phosphate (TCP) or Nano-hydroxyapatite (HA).

4. A cell-scaffold complex according to the claim 1, characterized in that the nanofibrous layer is produced from one or a combination of chitosan, collagen and polyvinyl alcohol.

5. A cell-scaffold complex according to the claim 4, characterized in that the mass ratio between collagen and chitosan is 1 to 5:1.

6. A cell-scaffold complex according to the claim 4, characterized in that the mass ratio between polyvinyl alcohol and chitosan is 1 to 5:1.

7. A cell-scaffold complex according to the claim 1, characterized in that the cell suspension comprises cell culture medium, serum and autologous cells; the mass ratio between cell culture medium and serum is in the range of 2 to 6:1, and the cell concentration is in the range of 10 4 /ml to 10 6 /ml; wherein the cells are osteoblast, corneal stromal cells or adipose-derived stem cells.

8. A 3D printing fabrication method of a cell-scaffold complex according to any preceding claims, characterized in that comprising the following steps: - 10 Step 1, forming a cell biological scaffold: blending raw materials of the cell biological scaffold evenly; producing a porous biological scaffold resemble with actual defects of tissues or organs by processing the blended raw materials layer by layer with scaffold printing head of bio-printer; Step 2, forming nanofibrous layers: dissolving nanofibrous layer material into deionized water at a certain ratio to produce electro spinning solution; spraying the electro spinning solution onto each layer or into gaps between layers by electrostatic spinning nozzle to form nanofibrous layers on the biological scaffold in a layer-by-layer manner; Step 3, cell printing: spraying the cell suspension on the nanofibrous layer by cell printing head and forming the cell-scaffold complex by adding up layers.

9. A 3D printing fabrication method of a cell-scaffold complex according to the claim 8, characterized in that adjacent pores inside the porous biological scaffold are connected with each other.

10. A 3D printing fabrication method of a cell-scaffold complex according to the claim 9, characterized in that the mass concentration of the electro spinning solution is in the range of 6% to 10%, and wherein the percentage of chitosan in the electro spinning solution is in the range of 0% to 5%, and the percentage of collagen in the electro spinning solution is in the range of 0% to 8% and the percentage of polyvinyl alcohol in the electro spinning solution is in the range of 0% to 10%.