阅读说明：本技术 一种仿生骨膜、骨膜-骨替代物及制备方法 (Bionic periosteum, periosteum-bone substitute and preparation method ) 是由 李斌 韩凤选 余颖康 于 2020-05-09 设计创作，主要内容包括：本发明要求保护一种用于大段骨缺损修复的仿生骨膜、骨膜-骨替代物及制备方法。所述的仿生骨膜是在聚二甲基硅氧烷表面培养成骨前体细胞至形成细胞片后,再脱除细胞获得细胞外基质(ECM)片。将仿生骨膜缠绕在可降解的骨支架材料表面即形成骨膜-骨替代物。本发明所制备的成骨前体细胞来源的ECM片不仅具有生物活性,而且在体内外均表现出骨诱导作用。此外,该ECM片对骨髓间充质干细胞具有趋化作用。通过结合细胞来源的ECM片与可降解骨支架材料,可改善目前临床上用于修复大面积节段性骨缺损的诱导膜技术,制备方法简单,可作为优异的骨膜-骨替代材料。(The invention claims a bionic periosteum, a periosteum-bone substitute and a preparation method for repairing large-section bone defects. The bionic periosteum is obtained by culturing osteogenic precursor cells on the surface of polydimethylsiloxane to form a cell sheet, and then removing the cells to obtain an extracellular matrix (ECM) sheet. Winding the bionic periosteum on the surface of the degradable bone scaffold material to form the periosteum-bone substitute. The ECM sheet derived from the osteoblast precursor cells prepared by the invention not only has biological activity, but also shows osteoinduction in vitro and in vivo. In addition, the ECM sheet has chemotactic effect on bone marrow mesenchymal stem cells. By combining the ECM sheet with cell source and the degradable bone scaffold material, the current induced membrane technology clinically used for repairing large-area segmental bone defect can be improved, the preparation method is simple, and the material can be used as an excellent periosteum-bone substitute material.)

1. A bionic periosteum is characterized in that an ECM sheet is obtained by culturing osteogenic precursor cells on the surface of polydimethylsiloxane, growing and maturing the cells into a cell sheet and then removing the cells.

2. A periosteum-bone substitute, characterized in that it consists of the biomimetic periosteum according to claim 1 and a bone scaffold material.

3. The periosteal-bone substitute according to claim 2, characterized in that said biomimetic periosteum is wound on the surface of a bone scaffold.

4. Periosteal-bone substitute according to claim 2 or 3, characterized in that said bone scaffold material is a degradable material.

5. Periosteal-bone substitute according to claim 2 or 3, characterized in that said bone scaffold material is selected from the group consisting of cylindrical, massive or irregular morphology.

6. The periosteum-bone substitute according to claim 2 or 3, characterized in that the biomimetic periosteum is present in a mass percentage of 0.01-10% of the periosteum-bone substitute.

7. The periosteal-bone substitute according to claim 2 or 3, characterized in that said bone scaffold material is selected from methacrylic anhydrified gelatin gel, sodium alginate hydrogel.

8. A method of making a biomimetic periosteum according to claim 1, comprising the steps of:

(1) preparing polydimethylsiloxane, carrying out plasma treatment on the polydimethylsiloxane, sterilizing, and adding fibronectin or gelatin for incubation;

(2) inoculating osteogenic precursor cells on the polydimethylsiloxane obtained in the step (1) for cell culture to obtain a cell sheet;

(3) after washing the cultured cell sheet with PBS, Triton-X100 and NH were used₄And (3) carrying out cell removal treatment on the cell sheet by using the OH mixed solution, then digesting residual DNA and RNA by using DNase and RNAse, and finally separating the ECM sheet from the polydimethylsiloxane.

9. The method according to claim 8, wherein in the step (2), when the cells grow to more than 80% of the surface of the polydimethylsiloxane, the ascorbic acid is added into the culture medium, and the culture is continued until the cell sheet is mature.

10. The method according to claim 8, wherein in step (2), the osteogenic precursor cells are selected from the group consisting of MC3T3-E1, mesenchymal stem cells of bone marrow, and adipose mesenchymal stem cells.

11. Use of a biomimetic periost according to claim 1 or a periost-bone substitute according to claim 2 for the preparation of a medical material for repairing a bone defect.

Technical Field

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

Background

The repair of large bone defects caused by severe wounds, tumors or infections is still a great challenge in orthopedics, and delayed healing or even non-healing often occurs in the repair process. It is well known that defects, when they exceed critical dimensions, cannot be completely repaired by their healing process.

Periosteal integrity is critical for fracture healing and bone regeneration. Periosteum is a thin tissue membrane covering the outer surface of bone, and delivers 70-80% of blood supply to cortical bone, providing osteoblasts, precursor cells and periosteal stem cells, playing an important role in bone formation and regeneration. Periosteum can promote healing by a variety of biological processes, such as cell proliferation and differentiation, or by paracrine signals that can recruit and activate host osteoprogenitor cells. Thus, if an artificial periosteum could perform similar functions to a natural periosteum, it would be helpful to repair a large bone defect.

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

Disclosure of Invention

The invention aims to provide a bionic periosteum, a periosteum-bone substitute and a preparation method, which can be used for repairing large-section bone defects.

In order to solve the technical problems, the invention provides a bionic periosteum-bone substitute which can effectively repair large-section bone defects. Wherein the prepared ECM sheet plays a role similar to periosteum, promotes the adhesion, migration and proliferation of peripheral periosteum cells, recruits stem cells to rapidly regenerate periosteum and bones at the defect, and the bone scaffold material can provide temporary support, avoid invasion of fibrous tissues and support the growth and osteogenesis of the bionic periosteum and the peripheral cells.

In order to achieve the above objects, a first aspect of the present invention provides a biomimetic periosteum for repairing a large-sized bone defect, which is obtained by culturing osteogenic precursor cells on a surface of Polydimethylsiloxane (PDMS), growing the cells into cell sheets, and then removing the cells to obtain ECM sheets.

In the preferred technical scheme of the invention, the ECM sheet of the bionic periosteum is osteoblast precursor cells, bone marrow mesenchymal stem cells, adipose-derived stem cells and the like, and the thickness of the ECM sheet is 10-50 mu m.

In a second aspect, the invention provides a periosteal-bone substitute consisting of an osteogenic precursor cell-derived ECM sheet 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 invention, the bone scaffold material is selected from but not limited to biomaterials such as methacrylic anhydride gelatin gel or sodium alginate hydrogel, and the biomaterials have good biocompatibility and degradability.

The periosteum-bone substitute can promote bone regeneration by the function of similar periosteum of an ECM sheet with biological activity and osteogenesis induction activity, and can be used as an excellent bone repair material for bone tissue engineering after being combined with a degradable bone scaffold material with a mechanical supporting function.

Periosteum-bone substitute can be constructed by wrapping prepared ECM sheet on the surface of bone scaffold material. Wherein the bionic periosteum accounts for 0.01-10% of the periosteum-bone substitute by mass percent.

The bone scaffold material can be methacrylic acid anhydridized gelatin gel or sodium alginate hydrogel, and other degradable bone scaffold materials known by the technical personnel in the field can be selected. The method for preparing methacrylic anhydrified gelatin (GelMA) gel is as follows: firstly, preparing GelMA solution, adding a photoinitiator (phenyl-2, 4, 6-trimethyl lithium benzoylphosphonate), injecting the solution into a quartz mould, and irradiating by using a blue light source flashlight with the wavelength of 365nm to obtain GelMA hydrogel. The method for preparing the sodium alginate hydrogel comprises the following steps: respectively preparing a sodium alginate solution and a calcium chloride solution, mixing the two solutions, and injecting the mixture into a quartz mold to obtain the sodium alginate hydrogel. The concentration of GelMA is preferably 0.1 w/v% -10 w/v%; the concentration of the sodium alginate is preferably 0.1 w/v% -2 w/v%. The shape of the bone scaffold material is preferably cylindrical or blocky, but may be irregular.

The third aspect of the invention provides a preparation method of a bionic periosteum, which comprises the following steps:

(1) preparing polydimethylsiloxane, placing the polydimethylsiloxane in a plasma cleaning machine, cleaning the polydimethylsiloxane in an oxygen atmosphere, sterilizing, and adding fibronectin or gelatin for incubation;

(2) inoculating osteogenic precursor cells on the polydimethylsiloxane obtained in the step (1) for cell culture to obtain a cell sheet;

(3) after washing the cultured cell sheet with PBS, Triton-X100 and NH were used₄And (3) carrying out acellular treatment on the cell sheet by using the OH mixed solution, then digesting residual DNA and RNA by using DNase and RNAse, and finally separating the ECM sheet from PDMS.

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

In the preferred embodiment of the present invention, in step (2), when the cells grow to more than 80%, preferably more than 90%, of the surface of the polydimethylsiloxane, ascorbic acid is added to the culture medium to promote the secretion of the extracellular matrix, and the culture is continued for 14 days until the cell sheet is mature, wherein the osteogenic precursor cells are MC3T3-E1 cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells, and the seeding density is 0.5 × 10⁴/cm²～2×10⁴/cm²The concentration of the ascorbic acid is 10-50 mu g/mL.

In the preferred technical scheme of the invention, in the step (3), the concentration of Triton-X is 0.1-0.25%, and NH is adopted₄The concentration of the OH solution is 10-50 mM, and the cell removing treatment time is 5-20 min; of DNases, e.g. DNase IThe concentration is 10-50U/mL, and the concentration of RNase, such as RNase A, is 0.5-2.5 μ L/mL.

In the preferable technical scheme of the invention, in the step (3), the cultured cell slice is washed by PBS and then is washed by Triton-X100 and NH₄The cells were decellularized with OH solution, then treated with DNaseI and RNase A at 37 ℃ for 2h to remove residual cellular DNA and RNA, and the ECM sheet was separated from PDMS and then stored at 4 ℃ for future use.

The fourth aspect of the invention provides the application of the bionic periosteum-bone substitute, which can be used for preparing medical materials for repairing bone defects, in particular for repairing large-section bone defects.

The invention cultures osteogenesis precursor cells on the surface of Polydimethylsiloxane (PDMS), after the cells are mature, the decellularization technology is utilized to remove the cells to obtain ECM sheets, and the ECM sheets are wound on the surface of GelMA hydrogel after photocrosslinking to construct periosteum-bone bionic bone substitute. The cell-derived ECM, due to its high bio-inducible activity and similar structure and composition to native periosteal ECM, will promote adhesion, migration and proliferation of surrounding periosteal cells and recruit stem cells to rapidly regenerate defective periosteum and bone. In addition, the bone scaffold material can play a temporary supporting role, avoid fibrous tissue invasion and support the growth and osteogenesis of bionic periosteum and surrounding cells.

In the invention, Polydimethylsiloxane (PDMS) can be a common commercial product or can be prepared; for example, the prepolymer and the curing agent in the Dow Corning 184 kit are mixed according to the mass ratio of 5: 1-30: 1, and curing to obtain polydimethylsiloxane, wherein the mass ratio of the prepolymer silicon elastomer base material to the curing agent is preferably 10: 1.

the invention has the advantages that:

(1) the invention prepares the ECM sheet (figure 1) from osteogenic precursor cells by a decellularization technology, and then the ECM sheet is wound and wrapped on the surface of hydrogel to prepare the bionic periosteum-bone which can effectively repair large-section bone defects;

(2) the ECM sheet can promote cell adhesion and proliferation, has a cell chemotaxis effect in vitro, shows a good osteogenesis induction effect in vivo and in vitro, can directly replace a tissue membrane generated by the induction of the existing membrane induction technology, plays a role similar to a periosteum, simplifies the operation process of the existing membrane induction technology, shortens the treatment time and has a very good application prospect;

(3) in the bionic periosteum-bone, the degradable bone scaffold material with good biocompatibility is used for replacing nondegradable polymethacrylate bone cement implanted in the membrane induction technology, so that the problems of temporary support and fibrous tissue ingrowth can be solved, and the step of taking out the bone cement in a second-stage operation required by the membrane induction technology can be omitted.

Drawings

FIG. 1A is an image of an ECM plate under an optical microscope; FIG. 1B is DAPI staining of ECM discs after decellularization; fig. 1C is an SEM image of the ECM sheet after decellularization.

Figure 2 appearance (2A) and SEM image (2B) of periosteum-bone substitute.

Fig. 3 is a picture of H & E staining of groups at week 12 of implantation of biomimetic periosteum-bone into a segmental bone defect in the radius of a rabbit, fig. 3A being an untreated group after defect; FIG. 3B is a hydrogel implant set; fig. 3C is the periosteum-bone substitute implant group. Wherein IM: implanting a material; NB: new bone; BM: bone marrow.

Detailed Description

The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. The conditions used in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments.

Introduction and summary

The present invention is illustrated by way of example and not by way of limitation. It should be noted that references to "an" or "one" embodiment in this disclosure are not necessarily to the same embodiment, but to at least one.

Various aspects of the invention are described below. It will be apparent, however, to one skilled in the art that the present invention may be practiced according to only some or all aspects of the present invention. For purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without specific details. In other instances, well-known features are omitted or simplified in order not to obscure the present invention.

Various operations will be described as multiple discrete steps in turn, and in a manner that is most helpful in understanding the present invention; however, the description in order should not be construed as to imply that these operations are necessarily order dependent.

Various embodiments will be described in terms of typical classes of reactants. It will be apparent to those skilled in the art that the present invention may be practiced using any number of different types of reactants, not just those provided herein for purposes of illustration. Furthermore, it will also be apparent that the invention is not limited to any particular hybrid example.

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