Cell microsphere induced bionic periosteum and preparation method thereof

Abstract

The invention provides a cell microsphere induced bionic periosteum and a preparation method thereof, and belongs to the technical field of bionic periostems.The preparation method comprises the steps that firstly, a PDA-PCL support is prepared, then cell microspheres obtained through a 3D culture plate are loaded on the support, PDSCs in the cell microspheres are dispersed out of an initial cell aggregate through cell culture, and the PDSCs in the cell microspheres are dispersed out of the initial cell aggregate; rapidly proliferating to form a layer of compact cell functional membrane, and preparing to obtain the cell microsphere induced bionic periosteum; the bionic periosteum can provide high-density stem cells, and can continuously secrete bioactive factors necessary for osteogenic differentiation and angiogenesis, so that the bionic periosteum has excellent osteogenic and angiogenesis promoting functions. According to the method provided by the invention, the bionic periosteum for simulating a natural bone environment is created, and a new bone defect repair strategy is provided: the bionic periosteum generated by expanding the PDSCs in the PDA-PCL stent provides a promising way for promoting bone regeneration treatment.

Description

Technical Field

[0001] The invention relates to the technical field of bionic periosteum, and in particular to a bionic periosteum induced by cell microspheres and a preparation method thereof. Background Art

[0002] The craniomaxillofacial bones and long bones of the limbs are susceptible to large-scale bone defects caused by trauma, infection, tumors and other iatrogenic factors. Traditional treatment methods mainly include autologous bone transplantation, allogeneic bone transplantation and allogeneic bone transplantation repair, but the source of autologous bone is limited and it causes additional trauma. Allogeneic bone and allogeneic bone are also limited in source, and have defects such as immune rejection and infectious diseases, which limit their widespread clinical application. In recent years, the rapid development of bone tissue engineering has brought hope for the clinical treatment of large-scale bone defects.

[0003] Existing bone tissue engineering scaffolds focus on constructing three-dimensional scaffolds to repair bone defects, but ignore the important role of periosteum in bone defect repair and regeneration; recent studies have shown that in the initial stage after autologous bone grafting, the contribution rate of periosteum to bone regeneration is about 70%, while current research designs mostly focus on mimicking the physical fiber structure of periosteum, while ignoring the biological activity of periosteum. Therefore, designing a bionic periosteum rich in stem cells and multiple active growth factors is the key to successfully repairing bone defects. Summary of the invention

[0004] The object of the present invention is to provide a cell microsphere-induced bionic periosteum and a preparation method thereof. The bionic periosteum prepared by the method provided by the present invention has a high density of stem cells and can continuously secrete bioactive factors necessary for osteogenic differentiation and angiogenesis, and has excellent osteogenesis and angiogenesis functions.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a bionic periosteum induced by cell microspheres, comprising the following steps:

[0007] (1) Using polycaprolactone as a raw material for 3D printing to obtain a polycaprolactone scaffold;

[0008] Immersing the polycaprolactone scaffold in a dopamine solution, stirring and post-treating in sequence to obtain a PDA-PCL scaffold;

[0009] (2) adding periosteal stem cells to a 3D culture plate for 3D culture to obtain cell microspheres;

[0010] (3) inoculating the cell microspheres obtained in step (2) onto the PDA-PCL scaffold obtained in step (1) for cell culture to obtain a bionic periosteum induced by the cell microspheres;

[0011] There is no particular order between step (1) and step (2).

[0012] Preferably, the molecular weight of the polycaprolactone in step (1) is 30 to 90 kDa.

[0013] Preferably, the conditions for 3D printing in step (1) are: the inner diameter of the nozzle is 250 μm, the moving speed is 8 to 12 mm / s, and the extrusion pressure is 160 to 220 kPa.

[0014] Preferably, in step (1), the polycaprolactone stent has a double-layer structure, and the thickness of the polycaprolactone stent is 200 to 800 μm.

[0015] Preferably, the concentration of dopamine in the dopamine solution in step (2) is 0.5-2 mg / mL; the dopamine solution is prepared by mixing dopamine with a Tris-HCl buffer solution having a pH value of 8.5 and a concentration of 1 mol / L.

[0016] Preferably, the periosteal stem cells in step (2) are skull periosteal stem cells from SD rats.

[0017] Preferably, the 3D culture in step (2) is as follows: adding the periosteal stem cells to a 3D culture plate at a cell density of 15,000 to 40,000 per well and culturing for 20 to 28 hours to obtain cell microspheres.

[0018] Preferably, the method for preparing the 3D culture plate in step (2) comprises the following steps: adding 2% bovine serum albumin solution into a 96-well plate with a U-shaped bottom, and letting it stand overnight to obtain a 3D culture plate.

[0019] Preferably, the cell culture time in step (3) is 6 to 8 days, and the culture medium used for the cell culture is 10% serum + 90% DMEM / F12.

[0020] The present invention also provides bionic periosteum induced by cell microspheres prepared by the preparation method described in the above technical solution.

[0021] The present invention provides a method for preparing a bionic periosteum induced by cell microspheres. First, a 3D-printed polycaprolactone (PCL) scaffold with a surface-adherent polydopamine (PDA) coating, namely a PDA-PCL scaffold, is prepared. Then, the cell microspheres obtained through a 3D culture plate are loaded on the scaffold. After cell culture, the PDSCs cells in the cell microspheres are dispersed from the initial cell aggregates, rapidly proliferate to form a dense cell functional membrane, and finally a bionic periosteum induced by cell microspheres is prepared; the bionic periosteum can provide high-density stem cells and can continuously secrete bioactive factors necessary for osteogenic differentiation and angiogenesis, so that the bionic periosteum has excellent osteogenic and angiogenic functions. The method provided by the present invention creates a bionic periosteum that simulates the natural bone environment and proposes a new strategy for bone defect repair: by expanding the bionic periosteum generated by PDSC cell microspheres in the PDA-PCL scaffold, it provides a promising way to promote bone regeneration therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of preparing the PDA-PCL scaffold in Example 1 of the present invention.

[0023] Figure 2 The scanning electron microscope images of the PCL scaffold and the PDA-PCL scaffold prepared in Example 1 of the present invention are shown in FIG. Figure 2 The scale bars in the first and second rows are 200 μm and 1 μm, respectively;

[0024] Figure 3 This is a diagram showing the results of energy spectrum analysis of the PCL scaffold and the PDA-PCL scaffold prepared in Example 1 of the present invention, wherein: Figure 3 Energy spectrum analysis was performed on three elements: C, O and N;

[0025] Figure 4 The microscopic image and hematoxylin-eosin staining (H&E) image of the cell microspheres prepared in Example 1 of the present invention, wherein: Figure 4 The middle left picture is a microscopic image. Figure 4 The middle right image is a hematoxylin-eosin (H&E) staining image;

[0026] Figure 5 This is a staining result of the cell microspheres prepared in Example 1 of the present invention after being inoculated on the PDA-PCL scaffold on the 7th day of cell culture. Figure 5 The first, second and third columns are the phalloidin staining images, the cell nucleus (DAPI) staining images and the merged images, respectively;

[0027] Figure 6The results of H&E staining of the bionic periosteum group and PDA-PCL group induced by the cell microspheres of the present invention were obtained from the subcutaneous tissue of the back of rats at the second week after implantation, wherein: Figure 6 The area indicated by the red arrow in the middle is the new blood vessel;

[0028] Figure 7 The Micro-CT results of the evaluation of new bone formation at the skull bone defect site in the control group, the cell microsphere-induced bionic periosteum group and the PDA-PCL group of the present invention at week 8; DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing a bionic periosteum induced by cell microspheres, comprising the following steps:

[0030] (1) Using polycaprolactone as a raw material for 3D printing to obtain a polycaprolactone scaffold;

[0031] Immersing the polycaprolactone scaffold in a dopamine solution, stirring and post-treating in sequence to obtain a PDA-PCL scaffold;

[0032] (2) adding periosteal stem cells to a 3D culture plate for 3D culture to obtain cell microspheres;

[0033] (3) inoculating the cell microspheres obtained in step (2) onto the PDA-PCL scaffold obtained in step (1) for cell culture to obtain a bionic periosteum induced by the cell microspheres;

[0034] There is no particular order between step (1) and step (2).

[0035] In the present invention, unless otherwise specified, the raw materials used are conventional commercial products in the art.

[0036] The present invention uses polycaprolactone as a raw material for 3D printing to obtain a polycaprolactone stent.

[0037] In the present invention, the molecular weight of the polycaprolactone is preferably 30 to 90 kDa, more preferably 40 to 80 kDa, and further preferably 60 kDa. The present invention uses polycaprolactone as a raw material to prepare polycaprolactone, and utilizes its degradability and non-toxic degradation products to improve the biocompatibility of the prepared bionic periosteum, and controls the molecular weight of polycaprolactone within the above range to improve the toughness, ductility and other mechanical properties of the bionic periosteum.

[0038] In the present invention, the conditions for 3D printing are preferably: the inner diameter of the nozzle is 250 μm, the moving speed is 8-12 mm / s, and the extrusion pressure is 160-220 kPa, and more preferably: the inner diameter of the nozzle is 250 μm, the moving speed is 10 mm / s, and the extrusion pressure is 200 kPa. The present invention controls the conditions for 3D printing within the above range to achieve continuous filament production and a suitable and uniform diameter of the filament.

[0039] In the present invention, the polycaprolactone stent preferably has a double-layer structure; the thickness of the polycaprolactone stent is preferably 200-800 μm, more preferably 300-700 μm. The present invention controls the thickness of the polycaprolactone stent within the above range to improve the toughness and ductility of the polycaprolactone stent.

[0040] After obtaining the polycaprolactone stent, the present invention immerses the polycaprolactone stent in a dopamine solution, and sequentially performs stirring and post-treatment to obtain a PDA-PCL stent.

[0041] In the present invention, the concentration of dopamine in the dopamine solution is preferably 0.5-2 mg / mL, more preferably 1 mg / mL; the dopamine solution is preferably prepared from dopamine and a Tris-HCl buffer solution with a pH value of 8.5 and a concentration of 1 mol / L.

[0042] In the present invention, the stirring temperature is room temperature; the stirring time is 20 to 28 hours. The present invention forms a dopamine coating (PDA) on the surface of the polycaprolactone scaffold by stirring, thereby reducing the hydrophobicity of the polycaprolactone scaffold and enhancing the adhesion, proliferation and growth of cells on the polycaprolactone scaffold, which is beneficial to the subsequent preparation of a bionic periosteum with excellent bone repair performance.

[0043] In the present invention, the post-treatment preferably includes sequentially performing pure water washing and vacuum drying.

[0044] The present invention has no particular limitation on the number of times of washing with pure water, and the dopamine adhering to the PDA-PCL stent can be removed. The present invention has no particular limitation on the method of vacuum drying, and the residual solvent can be removed.

[0045] The present invention adds periosteal stem cells to a 3D culture plate for 3D culture to obtain cell microspheres.

[0046] In the present invention, the periosteal stem cells are preferably skull periosteal stem cells of SD rats.

[0047] In the present invention, the method for preparing the periosteal stem cells is preferably as follows: according to the basic surgical operation specifications, the skull periosteum of 4-week-old SD rats is scraped off with a periosteal stripper, and placed in a 2% collagenase P solution for digestion for 4 hours; then complete cell culture medium (DMEM / F12 containing 10% fetal bovine serum) is added to terminate the digestion, and centrifugation, resuspending and plating are performed in sequence, and then placed in a 37°C, 20% carbon dioxide, humidified cell culture incubator for culture to obtain periosteal stem cells (abbreviated as PDSCs). Bone marrow mesenchymal stem cells (BMSCs) have the problems of easy aging and decreased cell stemness during in vitro culture. Compared with BMSCs as seed cells, the PDSCs selected in the present invention as the seed cells of the bionic periosteum have stronger activity and the characteristics of maintaining cell stemness, and at the same time have stronger osteogenic ability, which is conducive to obtaining the bionic periosteum with excellent osteogenic and angiogenic functions.

[0048] In the present invention, the 3D culture is preferably: adding periosteal stem cells to a 3D culture plate at a cell density of 15,000 / well to 40,000 / well and culturing for 20 to 28 hours to obtain cell microspheres, more preferably adding periosteal stem cells to a 3D culture plate at a cell density of 25,000 / well and culturing for 20 to 28 hours to obtain cell microspheres.

[0049] In the present invention, the method for preparing the 3D culture plate preferably comprises the following steps: adding a 2% bovine serum albumin solution into a 96-well plate with a U-shaped bottom, and leaving it to stand overnight to obtain a 3D culture plate.

[0050] There is no particular order in which the PDA-PCL scaffold and cell microspheres are prepared.

[0051] After obtaining the PDA-PCL scaffold and the cell microspheres, the present invention inoculates the cell microspheres onto the PDA-PCL scaffold for cell culture to obtain the bionic periosteum induced by the cell microspheres.

[0052] In the present invention, the cell culture time is preferably 6 to 8 days; the culture medium used for the cell culture is preferably 10% serum + 90% DMEM / F12.

[0053] The present invention also provides bionic periosteum induced by cell microspheres prepared by the preparation method described in the above technical solution.

[0054] In the present invention, the cell microsphere-induced bionic periosteum preferably includes a PDA-PCL scaffold and a layer of dense cell functional membrane formed by proliferation of cell microspheres loaded on the PDA-PCL scaffold.

[0055] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] Example 1

[0057] A method for preparing a bionic periosteum induced by cell microspheres, comprising the following steps:

[0058] (1) First, PCL particles with a molecular weight of Mn of 60 kDa were added to the hopper of a 3D printer (3D-Bioplotter) and fully dissolved at 120°C for 30 minutes; after the time was reached, 3D printing was performed, and the condition parameters of 3D printing were set as follows: the inner diameter of the nozzle was 250 μm, the moving speed was 10 mm / s, and the extrusion pressure was 200 kPa; 3D printing was performed to obtain a polycaprolactone scaffold with a double-layer structure and a thickness of 500 μm, namely, a PCL scaffold;

[0059] Then, the PCL stent was placed in a dopamine solution with a concentration of 1 mg / mL, and magnetic stirring was performed for 24 hours at room temperature to promote dopamine to adhere to the surface of the PCL stent to form a polydopamine coating, and then the unadhered dopamine was washed with pure water, and the stent was dried under vacuum to obtain a PDA-PCL stent for standby use;

[0060] The preparation method of the dopamine solution is as follows: dissolving dopamine powder with a molecular weight of 18964 g / mol in a 1 mol / L Tris-HCl buffer solution with a pH value of 8.5 and mixing by magnetic stirring;

[0061] (2) According to the basic surgical operation specifications, the periosteum of the skull of 4-week-old SD rats was scraped off with a periosteal stripper and placed in a 2% collagenase P solution for digestion for 4 hours; then complete cell culture medium (DMEM / F12 containing 10% Taiwanese bovine serum) was added to terminate the digestion, and centrifugation, resuspending and plating were performed in sequence, and then placed in a 37°C, 20% carbon dioxide, humidified cell culture incubator for culture to obtain periosteal stem cells (PDSCs); the periosteal stem cells were added to a 3D culture plate at a cell density of 25,000 / well for 3D culture for 24 hours to form cell microspheres;

[0062] The preparation method of the 3D culture plate is as follows: 2% bovine serum albumin solution is added into a 96-well plate with a U-shaped bottom, and the plate is allowed to stand overnight to form a 96-well plate with low adhesion and suitable for cell sphere culture, namely a 3D culture plate;

[0063] (3) inoculating the cell microspheres obtained in step (2) onto the PDA-PCL scaffold obtained in step (1) and performing cell culture for 7 days, wherein a dense cell functional membrane adheres to and spreads on the PDA-PCL scaffold, thereby obtaining a bionic periosteum induced by the cell microspheres;

[0064] The culture medium used for cell culture in step (3) is preferably 10% serum + 90% DMEM / F12.

[0065] Figure 1 This is a flow chart of preparing the PDA-PCL scaffold in Example 1 of the present invention.

[0066] Figure 2 The scanning electron microscope images of the PCL scaffold and the PDA-PCL scaffold prepared in Example 1 of the present invention are shown in FIG. Figure 2 The scale bars in the first and second rows are 200 μm and 1 μm, respectively. Figure 2 It can be seen that the 3D printed PCL has a uniform pore size, and dopamine coating has no obvious change on the pore size of the stent. The local enlarged image shows that there are many protruding dopamine particles on the surface of the PDA-PCL stent, which significantly increases the surface roughness, while the surface of the PCL stent is smooth.

[0067] Figure 3 This is the energy spectrum analysis result diagram of the PCL scaffold and PDA-PCL scaffold prepared in Example 1 of the present invention, Figure 3 It can be seen that the main elements of the PCL scaffold and the PDA-PCL scaffold are C and O elements, while the PDA-PCL scaffold can also contain N elements, which further illustrates the success of the coating strategy.

[0068] Microscopic image and hematoxylin-eosin staining (H&E) of the cell microspheres prepared in Example 1, as shown in Figure 4 As shown, Figure 4 The middle left picture is a microscopic image. Figure 4 The middle right picture is a hematoxylin-eosin (H&E) staining. Figure 4 It can be seen that the cell microspheres prepared in Example 1 are spherical and have a complete morphology; H&E staining shows that the cells are evenly distributed, well connected, and the center of the sphere is dense.

[0069] The cell microspheres prepared in Example 1 were inoculated on the PDA-PCL scaffold, and the cells were cultured for 7 days. Phalloidin and cell nucleus (DAPI) immunostaining was performed on the 7th day of culture, and the staining results of the cell microspheres prepared in Example 1 after inoculation on the PDA-PCL scaffold on the 7th day of cell culture were obtained as shown in the figure. Figure 5 As shown, Figure 5The first, second and third columns are Phalloidin staining images, DAPI staining images and Merge images, respectively. Figure 5 The first row of the pictures are all back, and the second row are all front. Figure 5 It can be seen that on the seventh day, the cells were completely spread and grown on the PDA-PCL scaffold, enhancing the mutual connection and interaction between cells, as if a layer of cell membrane structure was formed.

[0070] In order to detect the osteogenic and angiogenic functions of the bionic periosteum induced by the cell microspheres prepared in Example 1, an in vivo animal experiment was conducted, and three groups were set up respectively, and the operation was as follows:

[0071] (1) Control group (abbreviated as Control): blank group, in which skull bone defect was created but no treatment was performed;

[0072] (2) Cell microsphere-induced biomimetic periosteum group (abbreviated as Sph / PDA-PCL): The cell microsphere-induced biomimetic periosteum prepared in Example 1 was implanted into the subcutaneous tissue of the back and the surface of the 5 mm circular bone defect of the skull of 4-week-old SD rats, respectively. In the second week after implantation, the subcutaneous tissue samples of the back were collected, embedded, sliced ​​and stained (H&E staining). In the eighth week after implantation, the SD rats were subjected to Micro-CT scanning to observe the new bone regeneration at the skull bone defect site;

[0073] (3) PDA-PCL group (abbreviated as PDA-PCL): The operation was performed in the same manner as the cell microsphere-induced bionic periosteum group in step (2) above, except that the PDA-PCL scaffold prepared in step (1) of Example 1 was implanted subcutaneously on the back of 4-week-old SD rats and on the surface of a 5 mm circular bone defect on the skull, respectively, as a positive control group.

[0074] The above-mentioned in vivo animal experiment detected the H&E staining results of the subcutaneous tissue of the back of rats in the second week of the cell microsphere-induced bionic periosteum group and the PDA-PCL group. Figure 6 As shown by Figure 6 It can be seen that compared with the PDA-PCL group, the H&E staining of the cell microsphere-induced bionic periosteum group showed the formation of more blood vessels, indicating that the bionic periosteum has a good angiogenic function.

[0075] Micro-CT results of the evaluation of new bone formation in the skull bone defect site in the control group, cell microsphere-induced bionic periosteum group and PDA-PCL group at 8 weeks are shown in the figure. Figure 7 As shown. Figure 7 It can be seen that compared with the control group and the PDA-PCL group, the cell microsphere-induced bionic periosteum group generated more new bone tissue, achieving a good effect of repairing bone defects.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a bionic periosteum induced by cell microspheres, characterized in that: The following steps are involved: (1) Using polycaprolactone as a raw material for 3D printing to obtain a polycaprolactone scaffold; Immersing the polycaprolactone scaffold in a dopamine solution, stirring and post-treating in sequence to obtain a PDA-PCL scaffold; (2) adding periosteal stem cells to a 3D culture plate for 3D culture to obtain cell microspheres; (3) inoculating the cell microspheres obtained in step (2) onto the PDA-PCL scaffold obtained in step (1) for cell culture to obtain a bionic periosteum induced by the cell microspheres; There is no particular order between step (1) and step (2).

2. The preparation method according to claim 1, characterized in that: The molecular weight of the polycaprolactone in the step (1) is 30 to 90 kDa.

3. The preparation method according to claim 1, characterized in that: The conditions for 3D printing in step (1) are: the inner diameter of the nozzle is 250 μm, the moving speed is 8 to 12 mm / s, and the extrusion pressure is 160 to 220 kPa.

4. The preparation method according to claim 1, characterized in that: In the step (1), the polycaprolactone stent has a double-layer structure, and the thickness of the polycaprolactone stent is 200 to 800 μm.

5. The preparation method according to claim 1, characterized in that: The concentration of dopamine in the dopamine solution in step (2) is 0.5-2 mg / mL; the dopamine solution is prepared by mixing dopamine with a Tris-HCl buffer solution having a pH value of 8.5 and a concentration of 1 mol / L.

6. The preparation method according to claim 1, characterized in that: The periosteal stem cells in step (2) are skull periosteal stem cells from SD rats.

7. The preparation method according to claim 1, characterized in that: The 3D culture in step (2) is as follows: adding the periosteal stem cells to a 3D culture plate at a cell density of 15,000 to 40,000 per well and culturing for 20 to 28 hours to obtain cell microspheres.

8. The preparation method according to claim 1 or 7, characterized in that: The method for preparing the 3D culture plate in step (2) comprises the following steps: adding a 2% bovine serum albumin solution into a 96-well plate with a U-shaped bottom, and letting it stand overnight to obtain a 3D culture plate.

9. The preparation method according to claim 1, characterized in that: The cell culture time in step (3) is 6 to 8 days, and the culture medium used for the cell culture is 10% serum + 90% DMEM / F12.

10. The bionic periosteum induced by the cell microspheres prepared by the preparation method according to any one of claims 1 to 9.

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