WO2021227533A1

WO2021227533A1 - Biomimetic periosteum, periosteum-bone substitute, and preparation method therefor

Info

Publication number: WO2021227533A1
Application number: PCT/CN2020/141135
Authority: WO
Inventors: 李斌; 韩凤选; 余颖康
Original assignee: 苏州大学
Priority date: 2020-05-09
Filing date: 2020-12-30
Publication date: 2021-11-18
Also published as: CN111518755A

Abstract

A biomimetic periosteum and a periosteum-bone substitute for repairing a large-segment bone defect and a preparation method therefor. The biomimetic periosteum is obtained by culturing osteoblast precursor cells on the surface of polydimethylsiloxane to form cell sheets, and then removing the cells to obtain an extracellular matrix (ECM) sheet. A periosteum-bone substitute can be formed by wrapping the biomimetic periosteum on the surface of a degradable bone scaffold material. The prepared ECM sheet derived from osteogenic precursor cells has biological activity, and also exhibits osteoinductive effects both in vivo and in vitro. Additionally, the ECM sheet has a chemotactic effect on bone marrow mesenchymal stem cells. By combining the cell-derived ECM sheet with a degradable bone scaffold material, current clinically used induction membrane technology for repairing large-area segmental bone defects can be improved. The preparation method is simple and the prepared periosteum-bone substitute can be used as an excellent periosteum-bone substitute material.

Description

Bionic periosteum, periosteum-bone substitute and preparation method

Technical field

The invention relates to the technical field of biomedical materials, in particular to a bionic periosteum, a periosteum-bone substitute for repairing a large segment of bone defect, and a preparation method.

Background technique

The repair of large bone defects caused by severe trauma, tumor or infection is still a big challenge in orthopedics, and the healing process is often delayed or even non-union. As we all know, when the defect exceeds the critical size, it cannot be completely repaired through its own healing process.

Periosteum integrity is essential for fracture healing and bone regeneration. Periosteum is a thin tissue membrane covering the outer surface of bones, which transports 70-80% of the blood supply to the cortical bone, provides osteoblasts, precursor cells and periosteal stem cells, and plays an important role in bone formation and regeneration. Periosteum can promote healing through a variety of biological processes, such as cell proliferation and differentiation, or through paracrine signals, which can recruit and activate host osteoprogenitor cells. Therefore, if artificial periosteum can perform similar functions to natural periosteum, it will help repair large bone defects.

Extracellular matrix (ECM) has great potential as an artificial periosteum. ECM is a network composed of proteins and proteoglycans, containing a large number of growth factors and other signal molecules. In addition, it provides adhesion sites for cell growth and provides structural support for the interaction of cells with adjacent substrates. In addition, ECM can provide a microenvironment that regulates cell behavior and function.

Summary of the invention

The purpose of the present invention is to provide a bionic periosteum, periosteum-bone substitute which can be used to repair large segment bone defects and a preparation method.

In order to solve the above technical problems, the present invention provides a bionic periosteal-bone substitute, which can effectively repair large bone defects. Among them, the prepared ECM sheet will play a role similar to the periosteum, promote the adhesion, migration and proliferation of surrounding periosteal cells, and recruit stem cells to quickly regenerate the defect's periosteum and bone, while the bone scaffold material can provide temporary support and avoid fibrous tissue invasion And support the growth and bone formation of the bionic periosteum and surrounding cells.

To achieve the above objective, the first aspect of the present invention provides a biomimetic periosteum for repairing large bone defects. By culturing osteoblast precursor cells on the surface of polydimethylsiloxane (PDMS), the cells grow and mature into After the cell sheet, the cells are removed to obtain an ECM sheet.

In the preferred technical scheme of the present invention, the ECM sheet of the biomimetic periosteum is osteoblast precursor cells, bone marrow mesenchymal stem cells, adipose-derived stem cells, etc., and the thickness is 10-50 μm.

The second aspect of the present invention provides a periosteal-bone substitute, which is composed of an ECM sheet derived from osteogenic precursor cells and a bone scaffold material. Preferably, the bionic periosteum is wound on the surface of the bone scaffold material.

In the preferred technical scheme of the present invention, the bone scaffold material is selected from but not limited to biological materials such as methacrylic anhydride gelatin gel or sodium alginate hydrogel, and they all have good biocompatibility and degradability.

The periosteum-bone substitute of the present invention plays a role similar to the periosteum to promote bone regeneration through the ECM sheet with biological activity and osteoinductive activity, and can be used as an excellent bone when combined with a mechanical support and a degradable bone scaffold material. Repair materials are used for bone tissue engineering.

Periosteum-bone substitutes can be prepared by wrapping the prepared ECM sheet on the surface of the bone scaffold material to construct a periosteal-bone substitute. Among them, the bionic periosteum accounts for 0.01-10% of the mass percentage of the periosteum-bone substitute.

The bone scaffold material can be methacrylic acid anhydride gelatin gel or sodium alginate hydrogel, of course, other degradable bone scaffold materials known to those skilled in the art can also be used. The method of preparing methacrylic anhydride gelatin (GelMA) gel is as follows: first prepare GelMA solution, add photoinitiator (phenyl-2,4,6-trimethylbenzoylphosphonate lithium), and inject the solution The quartz mold was irradiated with a flashlight with a blue light source with a wavelength of 365 nm to obtain GelMA hydrogel. The method of preparing sodium alginate hydrogel is as follows: separately preparing sodium alginate solution and calcium chloride solution, mixing the two and injecting them into a quartz mold to obtain sodium alginate hydrogel. The concentration of GelMA is preferably 0.1w/v%-10w/v%; the concentration of sodium alginate is preferably 0.1w/v%-2w/v%. The shape of the bone scaffold material is preferably cylindrical or blocky, but it can also be irregular.

The third aspect of the present invention provides a method for preparing the bionic periosteal membrane, which includes the following steps:

(1) Prepare polydimethylsiloxane, place polydimethylsiloxane in a plasma cleaning machine, make it clean in an oxygen atmosphere, after sterilization, add fibronectin or gelatin for incubation;

(2) Inoculating osteoblast precursor cells on the polydimethylsiloxane obtained in step (1) for cell culture to obtain cell sheets;

(3) After washing the cultured cell sheet with PBS, first _{decellularize the cell sheet with a mixed solution of Triton-X100 and NH 4} OH, then use DNase and RNase to digest the remaining DNA and RNA, and finally The ECM sheet is separated from the PDMS.

In the preferred technical scheme of the present invention, in step (1), the concentration of fibronectin is 10-20 μg/mL, the concentration of gelatin is 0.5%-2% wt/v, and the incubation time is 6-8h.

In the preferred technical solution of the present invention, in step (2), when the cells grow to more than 80% of the polydimethylsiloxane surface, preferably 90% or more, ascorbic acid is added to the culture medium to promote the secretion of extracellular matrix , Continue to culture for 14 days until the cell sheet matures. The osteoblast precursor cells are MC3T3-E1 cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells, the seeding density is 0.5×10 ⁴ /cm ² ～2×10 ⁴ /cm ² , and the concentration of ascorbic acid is 10～ 50μg/mL.

In the preferred technical scheme of the present invention, in step (3), the concentration of Triton-X is 0.1% to 0.25%, _{the concentration of the NH 4} OH solution is 10 to 50 mM, and the decellularization treatment time is 5 to 20 minutes; DNA enzymes such as DNase The concentration of I is 10-50U/mL, and the concentration of RNase, such as RNase A, is 0.5-2.5μL/mL.

In the preferred technical scheme of the present invention, in step (3), after the cultured cell sheet is washed with PBS, _{the cells are decellularized with Triton-X100 and NH 4} OH solution, and then treated with DNase I and RNase A at 37°C 2h to remove residual cell DNA and RNA, separate the ECM sheet from the PDMS, and then store it at 4°C for later use.

The fourth aspect of the present invention provides the use of the above-mentioned bionic periosteal-bone substitute, which can be used for the preparation of medical materials for repairing bone defects, especially for the preparation of medical materials for repairing large-segment bone defects.

The present invention cultivates osteoblast precursor cells on the surface of polydimethylsiloxane (PDMS), after the cells mature, the cells are removed by decellularization technology to obtain an ECM sheet and wrap it on the surface of the photocrosslinked GelMA hydrogel To construct a periosteal-bone bionic bone substitute. The cell-derived ECM has high biological inducing activity and has a similar structure and composition to the natural periosteum ECM, which will promote the adhesion, migration and proliferation of surrounding periosteal cells, and recruit stem cells to quickly regenerate the defective periosteum and bone. In addition, the bone scaffold material will act as a temporary support, avoid fibrous tissue invasion, and support the growth and osteogenesis of the bionic periosteum and surrounding cells.

In the present invention, the polydimethylsiloxane (PDMS) can be a common commercial product, or it can be prepared; for example, the mass ratio of the precursor and curing agent in the Dow Corning 184 kit is 5:1-30 :1 mixing and curing to prepare polydimethylsiloxane, preferably, the mass ratio of the precursor silicone elastomer substrate to the curing agent is 10:1.

The advantages of the present invention are:

(1) The present invention prepares an ECM sheet derived from osteogenic precursor cells by decellularization technology (Figure 1), and then wraps it on the surface of the hydrogel to prepare a bionic periosteum-bone, which can be effectively repaired Large bone defect;

(2) The ECM sheet of the present invention can not only promote cell adhesion and proliferation, but also has cell chemotaxis in vitro, but also exhibits good osteogenic induction effects both in vivo and in vitro. It can directly replace the existing membrane induction technology for induction. The generated tissue membrane plays a role similar to the periosteum, thus simplifying the operation process of the existing membrane induction technology and shortening the treatment time, which has a very good application prospect;

(3) In the biomimetic periosteum-bone of the present invention, the non-degradable polymethacrylate bone cement implanted in the membrane induction technology is replaced with a degradable and good biocompatibility bone scaffold material. On the one hand, it can play a role Temporary support and avoiding the problem of fibrous tissue ingrowth, on the other hand, it can save the step of removing the bone cement in the second stage of the membrane induction technique.

Description of the drawings

Fig. 1A is an image of an ECM sheet under an optical microscope; Fig. 1B is a DAPI staining of an ECM sheet after decellularization; Fig. 1C is an SEM image of an ECM sheet after decellularization.

Figure 2 Appearance (2A) and SEM image (2B) of the periosteal-bone substitute.

Figure 3 shows the H&E staining pictures of each group at the 12th week when the bionic periosteum-bone is implanted into the segmental bone defect of the rabbit radius. Figure 3A is the untreated group after the defect; Figure 3B is the hydrogel implantation group; Figure 3C shows the periosteal-bone substitute implantation group. Among them, IM: implant material; NB: new bone; BM: bone marrow.

Detailed ways

The above solution will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are used to illustrate the present invention and not to limit the scope of the present invention. The implementation conditions used in the examples can be further adjusted according to the conditions of specific manufacturers, and implementation conditions not specified are usually conditions in routine experiments.

Introduction and overview

The present invention is illustrated by way of examples rather than limitations. It should be noted that the "a" or "an" embodiment described in this disclosure does not necessarily refer to the same specific embodiment, but refers to at least one.

Hereinafter, various aspects of the present invention will be described. However, it is obvious to those skilled in the art that the present invention can be implemented according to only some or all aspects of the present invention. For the sake of illustration, specific numbers, materials and configurations are given herein to enable people to thoroughly understand the present invention. However, it will be obvious to those skilled in the art that the present invention can be implemented without specific details. In other instances, well-known features have been omitted or simplified in order not to obscure the present invention.

Various operations are described in sequence as a plurality of discrete steps, and are described in a manner that is most helpful for understanding the present invention; however, the sequential description should not be construed as implying that these operations necessarily depend on the sequence.

Various embodiments will be explained based on typical kinds of reactants. It will be obvious to those skilled in the art that the present invention can be implemented using any number of different kinds of reactants, not just those reactants given here for illustrative purposes. In addition, it will also be apparent that the present invention is not limited to any specific hybrid example.

In the present invention, the silicon elastomer substrate is a Dow Corning commercial product, MC3T3-E1 cells are a commercial product, and bone marrow mesenchymal stem cells and adipose mesenchymal stem cells are extracted from rat tissues.

Example 1:

(1) Mix the prepolymer and curing agent in the Dow Corning 184 kit according to a mass ratio of 10:1, and place them at 60°C for curing for 2 hours to prepare PDMS. The PDMS is placed in a plasma cleaning machine and cleaned in an oxygen atmosphere. Before cell culture, PDMS sterilizes the PDMS substrate with ultraviolet light, and then incubates with 20μg/mL fibronectin for 6-8h;

(2) Inoculate MC3T3-E1 cells on the hatched PDMS at a density of 0.5×10 ⁴ /cm ² , when the cells grow to 80% of the surface, add 20 μg/mL ascorbic acid to the medium and continue the culture;

(3) After 14 days, wash the cultured cell sheet 3 times with PBS, first use 0.25% Triton-X100 and 25mM NH ₄ OH solution to decellularize the cells for 10 minutes, then use 25U/mL DNase I and 1.5μL /mL RNase A is treated at 37°C for 2h to remove residual cellular DNA and RNA, and the prepared ECM slices are stored at 4°C for later use. The appearance of the ECM slices is shown in Figure 1 of the instruction manual;

(4) Add PBS to the GelMA solid sponge, then place it in a 37℃ water bath to dissolve to a transparent liquid, and then add a photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) After dissolution, a GelMA solution with a concentration of 5% is obtained. Inject the GelMA solution into a quartz mold, irradiate it with a blue light source flashlight with a wavelength of 365nm for 1 min, and obtain a GelMA column with a length of 10mm and a diameter of 10mm;

(5) Wrap the prepared ECM sheet on the surface of the GelMA column to construct a bionic periosteal-bone structure.

Example 2:

(1) The prepolymer and curing agent in the Dow Corning 184 kit are mixed according to a mass ratio of 20:1, then cured to prepare PDMS, and plasma treatment is performed. Before cell culture, UV sterilize the prepared PDMS, and then incubate it with gelatin solution for 8 hours;

(2) Inoculate the bone marrow mesenchymal stem cells on the hatched PDMS at a density of 2×10 ⁴ /cm ^{2. When the} cells grow to 90% of the surface, add 50 μg/mL ascorbic acid to the medium to promote extracellular Secretion of the matrix, continue to culture until the cell sheet matures;

(3) Wash the cultured cell sheet 3 times with PBS, first use 0.25% Triton-X100 and 50mM NH ₄ OH solution to decellularize the cells for 5 minutes, then use 50U/mL DNaseI and 2.5μL/mL RNase A Treated at 37°C for 2h to remove residual cell DNA and RNA, ECM tablets have good biocompatibility;

(4) Add PBS to the GelMA solid sponge, then place it in a 37℃ water bath to dissolve to a transparent liquid, and then add a photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) After dissolution, a GelMA solution with a concentration of 10% is obtained. Inject the GelMA solution into a quartz mold, and irradiate it with a blue light source flashlight with a wavelength of 405 nm for 1 min to obtain a GelMA column with a length of 15 mm and a diameter of 5 mm;

(5) Wrap the prepared ECM sheet on the surface of the GelMA column to construct a bionic periosteum-bone structure. The appearance and SEM image of the bionic periosteum-bone are shown in Figure 2. Compared with the control group and the pure GelMA group, the bionic periosteal-bone group has a good repair effect (Figure 3).

Example 3:

(1) The prepolymer and curing agent in the Dow Corning 184 kit are mixed according to a mass ratio of 5:1, and cured to prepare PDMS, which is treated by plasma. Before cell culture, UV sterilize the PDMS substrate, and then incubate with 10μg/mL fibronectin for 6-8h;

(2) Inoculate adipose-derived mesenchymal stem cells on the hatched PDMS at a density of 1×10 ⁴ /cm ^{2. When the} cells grow to 90% of the surface, add 40 μg/mL ascorbic acid to the medium to promote extracellular Secretion of the matrix, continue to culture until the cell sheet matures;

(3) Wash the cultured cell sheet 3 times with PBS, first use 0.1% Triton-X100 and 40mM NH ₄ OH solution to decellularize the cells for 20 minutes, then use 40U/mL DNase I and 2.0 μL/mL RNase A Treat it at 37°C for 2h to remove residual cellular DNA and RNA, and store the prepared ECM slice at 4°C for later use;

(4) Prepare a 2% sodium alginate solution and a 10mM calcium chloride solution, mix them and add them to the mold to prepare a hydrogel column with a diameter of 5mm and a height of 10mm;

(5) Wrap the prepared ECM sheet on the surface of the hydrogel column to construct a biomimetic periosteal-bone structure.

The specific embodiments described above are only preferred implementations of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements or replacements can be made. These improvements Or replacement should also be regarded as the protection scope of the present invention.

Claims

A bionic periosteum, which is characterized in that the ECM sheet is obtained by culturing osteogenic precursor cells on the surface of polydimethylsiloxane, and removing the cells after the cells grow and mature into cell sheets.
A periosteal-bone substitute, characterized in that it is composed of the bionic periosteal of claim 1 and a bone scaffold material.
The periosteal-bone substitute according to claim 2, wherein the bionic periosteum is wound on the surface of the bone scaffold.
The periosteal-bone substitute according to claim 2 or 3, wherein the bone scaffold material is a degradable material.
The periosteal-bone substitute according to claim 2 or 3, wherein the shape of the bone scaffold material is selected from a cylindrical shape, a block shape, or an irregular shape.
The periosteal-bone substitute according to claim 2 or 3, wherein the mass percentage of the bionic periosteum in the periosteal-bone substitute is 0.01-10%.
The periosteal-bone substitute according to claim 2 or 3, wherein the bone scaffold material is selected from the group consisting of methacrylic anhydride gelatin gel and sodium alginate hydrogel.
The method for preparing the bionic periosteum according to claim 1, comprising the following steps:

(1) Prepare polydimethylsiloxane, treat polydimethylsiloxane with plasma, and after sterilization, add fibronectin or gelatin for incubation;

(2) Inoculating osteoblast precursor cells on the polydimethylsiloxane obtained in step (1) for cell culture to obtain cell sheets;

(3) After washing the cultured cell sheet with PBS, first decellularize the cell sheet with a mixed solution of Triton-X100 and NH 4 OH, then use DNase and RNase to digest the remaining DNA and RNA, and finally The ECM sheet is separated from the polydimethylsiloxane.
The method according to claim 8, characterized in that, in step (2), when the cells grow to more than 80% of the surface of the polydimethylsiloxane, ascorbic acid is added to the medium, and the culture is continued until the cell sheet matures.
The method according to claim 8, wherein in step (2), the osteoblast precursor cells are selected from MC3T3-E1, bone marrow mesenchymal stem cells or adipose-derived mesenchymal stem cells.
The use of the bionic periosteum of claim 1 or the periosteal-bone substitute of claim 2 is used to prepare medical materials for repairing bone defects.