CN107890585B

CN107890585B - Composite ceramic bracket and preparation method thereof

Info

Publication number: CN107890585B
Application number: CN201711088223.8A
Authority: CN
Inventors: 沈利燕; 唐千; 徐华梓
Original assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Current assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Priority date: 2017-10-31
Filing date: 2017-10-31
Publication date: 2020-11-10
Anticipated expiration: 2037-10-31
Also published as: CN107890585A

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a composite ceramic support and a preparation method thereof, in particular to preparation of a multi-layer film modified porous double-phase ceramic support and application thereof as a bone defect repair material. The preparation method of the composite ceramic bracket provided by the invention comprises the following steps: and (2) sequentially soaking the porous ceramic support in the solution A, the solution C, the solution B and the solution D to obtain a double-layer multilayer film, repeating the steps at least once to obtain a plurality of double-layer multilayer films, soaking the double-layer multilayer films in the aqueous solution of a cross-linking agent for cross-linking for 30min to 24 hours, and fully cleaning the double-layer multilayer films in the washing solution to obtain the cross-linked composite ceramic support. The composite ceramic scaffold multilayer film provided by the invention has the advantages that the medicine acts on the wound in situ through the enrichment and riveting effects of the composite ceramic scaffold multilayer film, the osteogenic differentiation of stem cells is promoted, and the composite ceramic scaffold multilayer film has a great promotion effect on bone regeneration of osteoporosis patients.

Description

Composite ceramic bracket and preparation method thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a composite ceramic support and a preparation method thereof, in particular to preparation of a multi-layer film modified porous double-phase ceramic support and application thereof as a bone defect repair material.

Background

Bone defects caused by trauma, osteomyelitis, bone tumor excision and the like are often long in treatment process and uncertain in curative effect, and cause great pain to patients in both physiology and spirit. Currently, in clinical treatment of bone defects, autologous iliac bone transplantation and Ilizarov bone lengthening are mainly adopted. However, in the face of large-area bone defects, the amount of autologous bone cannot meet the requirements, and patients need to suffer from long-term pain and risk of infection due to an external fixing bracket according to the bone lengthening rule, so that artificial bone substitute materials are often required to be introduced. Among these, biphasic ceramic particles, as well as porous ceramic scaffolds, screws, etc., have been FDA approved for use in orthopedic surgery. However, the bone formation effect is not ideal due to the lack of effective bone induction effect when the materials such as ceramics and screws are simply implanted, and the effect is worse especially for patients suffering from basic diseases such as osteoporosis.

Layer-by-layer self-assembly (LBL) is a simple film-forming technique that can organically integrate multiple functional molecules. The material can improve the biological activity of an implant and endow the material with new characteristics under the condition of not changing the mechanical property of the material, and has wider and wider application in the field of orthopedic implantation. The technology can promote the adhesion and proliferation of cells by selecting the polyelectrolyte with higher biological affinity to construct the coating, and on the other hand, the multilayer film structure can better promote the enrichment of drugs/growth factors, and is a good platform for regulating physical, chemical and biological factors on the surface of the implant material. PICART and the like (Small, 2009, 5 (5): 598-608) utilize a layer-by-layer assembly technology to coat multiple layers of Polylysine (PLL) and Hyaluronic Acid (HA) on the surface of the porous ceramic scaffold, and simultaneously load Bone morphogenetic protein-2 (BMP-2) to construct the ceramic matrix composite scaffold, so that the subcutaneous ectopic Bone formation effect is good. However, the research shows that the osteogenesis activity of the composite scaffold is mainly from BMP-2, but not from multilayer films, and the cladding of the multilayer films on the ceramic scaffold does not have any advantages in vitro cell experiments and in vivo ectopic osteogenesis experiments. The use of BMP-2 protein is not only extraordinarily expensive, but also has great hidden danger and risk of causing inflammation.

Disclosure of Invention

In order to solve the above technical problems, the present invention is to construct a simple, safe and effective composite scaffold based on porous ceramic material for bone repair and regeneration treatment of large-area bone defects, in order to enhance the bioactivity of porous ceramic scaffolds and simultaneously avoid the high cost and risks of inflammation and carcinogenesis caused by using growth factors such as BMP.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a composite ceramic bracket comprises the steps of sequentially soaking a porous ceramic bracket in solution A, solution C, solution B and solution D to obtain a double-layer multilayer film, repeating the steps at least once to obtain a plurality of double-layer multilayer films, soaking the double-layer multilayer films in an aqueous solution of a cross-linking agent for cross-linking for 30min to 24 hours, and fully cleaning the double-layer multilayer films in a washing solution to obtain the cross-linked composite ceramic bracket;

the solution A is a polyelectrolyte solution with positive electricity, the concentration is 0.1-10mg/mL, and the pH value is 2.5-9.5; the solution B is a polyelectrolyte solution with negative electricity, the concentration is 0.1-10mg/mL, and the pH value is 2.5-5.0.

The porous ceramic scaffold is a porous scaffold with communicating holes, and the scaffold material comprises one or more of beta-TCP, hydroxyapatite and bioactive glass.

Wherein the solution A is one or more aqueous solutions of chitosan, modified positively charged hyaluronic acid and polylysine.

And the washing solution C and the washing solution D are both deionized water. The washing solution can be water, buffer solution or salt solution.

Wherein the solution B is one or more aqueous solutions of hyaluronic acid, alginic acid, heparin, chondroitin sulfate and soluble salts thereof.

Wherein the cross-linking agent is one or more of carbodiimide cross-linking agent, genipin, glutaraldehyde and succinimide.

Wherein, the crosslinked composite ceramic bracket is further soaked in a drug solution to load drugs for promoting bone repair so as to enhance the curative effect.

Wherein the medicine solution is one or more of calcitriol, bisphosphonate, strontium ranelate and dexamethasone.

The solvent of the drug solution is ethanol, but the solvent is not limited to ethanol, and a solvent capable of dissolving is all possible.

The composite ceramic bracket prepared by the preparation method.

The natural polysaccharide such as hyaluronic acid and chitosan and the natural polypeptide such as polylysine are all widely existing natural macromolecules, have biodegradability and good biocompatibility, and have the effects of promoting the proliferation, adhesion and differentiation of mesenchymal stem cells.

Calcitriol is an activated form of vitamin D in vivo, is an oral medicament for treating osteoporosis, and can act on enterocytes in small intestine to promote absorption of calcium ions by small intestine and maintain balance of bone formation and bone absorption of the whole body after oral administration. The direct application of calcitriol in the focus part is expected to act on mesenchymal stem cells to induce osteogenic differentiation of the mesenchymal stem cells, so that the bone regeneration of the defect part is accelerated.

Bisphosphonates are a class of drugs used in various bone disorders and calcium metabolic diseases. Generally, the medicine can be absorbed by oral administration, and can be specifically combined with hydroxyapatite in bone to inhibit osteoclast activity, thereby inhibiting bone absorption. Also, by in situ application at the focal site, it is possible to directly act on osteoclasts and other osteocytes at the defect site, thereby promoting osteogenesis. In-situ application of the traditional oral medicines such as strontium ranelate, dexamethasone and the like is expected to locally promote bone repair, and simultaneously, toxic and side effects caused by systemic medication are avoided.

On the basis of keeping the osteogenic activity of the original ceramic scaffold, the composite ceramic scaffold introduces the induction action of polysaccharide/polypeptide biomacromolecules on the adhesion, proliferation and differentiation of mesenchymal stem cells, has good bone regeneration induction at the defect part, and is suitable for repairing the defect of a large bone; by introducing traditional medicines for treating osteoporosis and calcium metabolism diseases, the bone regeneration can be further accelerated, and the traditional Chinese medicine composition has a good curative effect even under the condition of suffering from systemic skeletal system diseases such as osteoporosis.

The composite ceramic bracket provided by the invention has the following advantages and characteristics:

1. the invention achieves the treatment effect of addition by simply modifying the existing materials which are clinically popularized and carrying the medicines, and the hyaluronic acid, the chitosan and the like which construct the coating are all biological materials which can be widely obtained, are economical and practical, and have economic advantages.

2. According to the invention, the multi-layer membrane with a porous structure is formed by regulating and controlling the change of pH in the layer-by-layer self-assembly process, and the porous rough membrane surface is more favorable for cell adhesion and proliferation on the premise of not influencing the ion release of the material.

3. Calcitriol and other medicaments are FDA approved medicaments for treating clinical osteoporosis patients, and are safe and reliable in application. The invention makes the medicine act on the wound in situ through the enrichment and riveting functions of the multilayer film, promotes the osteogenic differentiation of stem cells, and has great advantages for the bone regeneration of osteoporosis patients.

Drawings

Fig. 1 is a schematic view of the preparation process of the composite ceramic scaffold of the present invention, wherein a is the ceramic scaffold.

FIG. 2 is a graph showing the in vitro release curves of calcium ions of examples 1 to 8 and comparative examples 1 to 2. Wherein 1 to 8 represent examples 1 to 8 and 9 to 10 represent comparative examples 1 to 2.

FIG. 3a is a topographical map of the ceramic surface of example 1, with a 5 micron scale.

FIG. 3b is a surface topography of the ceramic of comparative example 1 with a 5 micron scale.

FIG. 4a is a graph showing the growth behavior of normal mesenchymal stem cells on a composite ceramic scaffold in examples 1 to 8 and comparative examples 1 to 2. Wherein 1 to 8 represent examples 1 to 8 and 9 to 10 represent comparative examples 1 to 2.

FIG. 4b is a graph showing the growth behavior of the osteoporosis mesenchymal stem cells on the composite ceramic scaffold in examples 1-8 and comparative examples 1-2. Wherein 1 to 8 represent examples 1 to 8 and 9 to 10 represent comparative examples 1 to 2.

Detailed Description

The invention will now be further illustrated by reference to the following examples:

the present invention is embodied as follows.

The invention is further described with reference to the accompanying drawings and detailed description.

The composite scaffold is prepared by the method shown in figure 1, a ceramic scaffold substrate is firstly soaked in the solution A and stands for 10min to adsorb a layer of molecules A, washed off to remove substances A which are not firmly adsorbed in the washing solution, then soaked in the solution B and soaked for 10min to adsorb a layer of molecules B, washed off to remove substances B which are not firmly adsorbed in the washing solution, a double-layer multilayer film is obtained, and the steps are repeated to prepare the required multilayer film.

In the embodiment, the adopted support base materials are round blocks with the diameter of 8mm and the thickness of 2mm, the porous structures are communicated holes, the specific parameters are 60 percent of hydroxyapatite, 40 percent of beta-tricalcium phosphate, the porosity of the support is 75-85 percent, and the average pore diameter is 200 mu m. Other conventional porous ceramic scaffolds can achieve similar effects.

The A, B solutions used in the examples were all prepared in deionized water, adjusted to the desired pH by adding dropwise 1M sodium hydroxide solution or 1N hydrochloric acid solution, the wash being deionized water. The solvent of the drug solution used in the examples was ethanol, and after soaking the drug solution, it was washed in ethanol and dried in a vacuum oven to remove residual ethanol.

Example 1 a chitosan solution (solution a, concentration 1mg/mL, ph4.0) and a sodium hyaluronate solution (solution B, concentration 3mg/mL, ph3.0) were alternately deposited on the surface of a ceramic stent to obtain a 10-layered multilayer film, and the stent coated with the multilayer film was immersed in a carbodiimide-succinimide solution to crosslink for 24 hours, thoroughly washed, dried, and sterilized for use.

Example 2. a polylysine solution (solution a, concentration 0.1mg/mL, ph9.5) and a sodium heparin solution (solution B, concentration 5mg/mL, ph5.0) were alternately deposited on the surface of a ceramic stent to obtain a 20-layer multilayer film, and the stent coated with the multilayer film was immersed in a genipin solution to be crosslinked for 12 hours, sufficiently washed, dried, and sterilized for use.

Example 3. a ceramic stent surface was alternately deposited with an aminated hyaluronic acid solution (solution a, concentration 5mg/mL, ph8.0) and a chondroitin sulfate solution (solution B, concentration 0.1mg/mL, ph2.5) to obtain a 5-layer multilayer film, and the stent coated with the multilayer film was immersed in a glutaraldehyde solution for crosslinking for 0.5 hour, thoroughly washed, dried, and sterilized for use.

Example 4a chitosan solution (solution a, concentration 10mg/mL, ph2.5) and a potassium alginate solution (solution B, concentration 10mg/mL, ph3.0) were alternately deposited on the surface of a ceramic stent to obtain a 50-layer multilayer film, the stent coated with the multilayer film was immersed in a carbodiimide solution for crosslinking for 18 hours, thoroughly washed, dried, and sterilized for use.

Example 5 a chitosan solution (solution a, concentration 1mg/mL, ph4.0) and a sodium hyaluronate solution (solution B, concentration 3mg/mL, ph3.0) were alternately deposited on the surface of a ceramic stent to obtain a 10-bilayer multilayer film, and the multilayer film-coated stent was immersed in a carbodiimide-succinimide solution to crosslink for 24 hours. Cleaning, drying, soaking in medicinal solution (dexamethasone alcoholic solution), cleaning, drying, and sterilizing.

Example 6. polylysine solution (solution a, concentration 0.1mg/mL, ph9.5) and sodium heparin solution (solution B, concentration 5mg/mL, ph5.0) were alternately deposited on the surface of a ceramic stent to give a 20-layer multilayer film, and the multilayer film-coated stent was immersed in genipin solution and crosslinked for 12 hours. Cleaning, drying, soaking in medicinal solution (strontium ranelate alcoholic solution), cleaning, drying, and sterilizing.

Example 7. a ceramic stent surface was alternately deposited with an aminated hyaluronic acid solution (solution a, concentration 5mg/mL, ph8.0) and a chondroitin sulfate solution (solution B, concentration 0.1mg/mL, ph2.5) to give a 5-layer multilayer film, and the multilayer film-coated stent was immersed in a glutaraldehyde solution to crosslink for 0.5 hour. Cleaning, drying, soaking in medicinal solution (alcoholic solution of alendronate sodium), cleaning, drying, and sterilizing.

Example 8 a chitosan solution (solution a, concentration 10mg/mL, ph2.5) and a potassium alginate solution (solution B, concentration 10mg/mL, ph3.0) were alternately deposited on the surface of a ceramic stent to obtain a 50-layer multilayer film, and the multilayer film-coated stent was immersed in a carbodiimide solution for crosslinking for 18 hours. Cleaning, drying, soaking in medicinal solution (alcoholic solution of calcitriol), cleaning, drying, and sterilizing.

Comparative example 1 referring to the reference, polylysine (solution a, concentration 0.5mg/mL, ph7.4) and sodium hyaluronate (solution B, concentration 1mg/mL, ph7.4) were alternately deposited on the surface of a ceramic stent to obtain a 24-layer multilayer film, and the stent coated with the multilayer film was immersed in a carbodiimide-succinimide solution for crosslinking for 18 hours, thoroughly washed, dried, and sterilized for use.

Comparative example 2 ceramic scaffolds, without surface treatment, were sterilized for use.

The above examples 1-8 are experimental groups and comparative examples 1-2 are control groups, and the following tests were performed:

1. calcium ion in vitro release behavior detection

The obtained composite ceramic scaffold was placed in 100mL of Phosphate Buffered Saline (PBS), sealed and placed in a 37 ℃ shaking table, 0.5mL of the released PBS solution was taken at 1 day, 3 days, 7 days, 12 days, 20 days, and 35 days, respectively, and the calcium ion concentration was measured by atomic absorption spectroscopy, and the obtained calcium ion release curves of the different examples are shown in fig. 2.

As can be seen from fig. 2, the calcium ion release behavior of examples 1 to 8 is similar to that of comparative example 2 (unmodified ceramic stent), whereas the release of calcium ions of comparative example 1 is significantly slowed. According to the invention, a porous multilayer film structure (figure 3a) is obtained by reducing the pH value of polyanion (solution B) in the layer-by-layer assembly process, so that calcium ions in the obtained composite ceramic scaffold can be rapidly released, and the osteogenic activity of mesenchymal stem cells on the surface and around the material can be greatly promoted.

2. Mesenchymal stem cell adhesion proliferation assay

Firstly, carrying out osteoporosis rat modeling: healthy 200-220g SD female rats are selected for bilateral ovariectomy, and a postmenopausal osteoporosis model (OVX for short) is established after the animals are raised for three months.

Taking an osteoporosis SD rat which is successfully molded and a normal SD rat with the same week age, taking bilateral femoral shafts under an aseptic environment after being killed by a pentobarbital sodium anesthesia, and extracting Mesenchymal Stem Cells (MSC) by a full bone marrow adherence method. Obtaining normal rat mesenchymal stem cells BMSC and osteoporosis rat mesenchymal stem cells OVX-BMSC.

BMSC and OVX-BMSC are respectively inoculated on the obtained composite ceramic scaffold, and the adhesion proliferation condition of cells is measured by a CCK-8 kit after 3 days, 7 days or 14 days of inoculation. The test results are shown in fig. 4.

As can be seen from fig. 4a, normal mesenchymal stem cells are more easily adhered to the multilayer films of the experimental group, and proliferate more rapidly, which can be attributed to the porous structures of the multilayer films, which not only facilitate the normal release of calcium ions, but also have strong promoting and inducing effects on the adhesion and proliferation of mesenchymal stem cells. The smooth multilayer film of example 9 showed less cell adhesion and proliferation than the untreated ceramic scaffold, which is also consistent with literature reports.

Fig. 4b shows the adhesion proliferation of OVX-BMSC on different composite ceramic scaffolds, which shows that the proliferation rate of mesenchymal stem cells extracted from osteoporosis rats is obviously reduced, but the existence of the porous multilayer film can also effectively promote the adhesion and proliferation thereof, and meanwhile, the introduction of the osteoporosis drug further promotes the proliferation of OVX-BMSC.

3. In vivo bone defect regeneration experimental detection

6 osteoporotic rats were taken out of each group, circular quantitative defects (defect range incapable of self-repair) of 8mm in diameter were drilled out on the surface of the skull of the rats by a slow dental annular drill, and the obtained scaffold material was filled. The wound is then closed by layer closure. At the middle (6 weeks) and later (12 weeks) of bone repair, 3 rats were sacrificed, respectively, the cranium was removed, subjected to micro-CT scanning and three-dimensional reconstruction, and the osteogenesis amount was calculated. The results are shown in Table 2.

The ceramic scaffolds coated with the porous multilayer film of the present invention (examples 1-4) had better bone formation in both the middle and late stages than the unmodified ceramic scaffold (comparative example 2) in osteoporotic rats, with twice as much new bone formation as the latter; further, the traditional oral osteoporosis drugs (examples 5-8) were introduced into the composite ceramic scaffold, and the new bone mass was further increased, which greatly exceeded the promotion effect of drug addition on cell proliferation in previous cell experiments, and it was shown that the drugs more act on osteogenic differentiation of mesenchymal stem cells of rats with osteoporosis.

Table 1: bone formation amount of 6 weeks and 12 weeks after the osteoporosis rat is implanted into the ceramic composite scaffold

(the ratio of the new bone volume to the total volume of the implanted ceramic scaffold (totalvolume))

The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the present invention should not be limited by the disclosure of the preferred embodiments. Therefore, it is intended that all equivalents and modifications which do not depart from the spirit of the invention disclosed herein are deemed to be within the scope of the invention.

Claims

1. A preparation method of a composite ceramic support is characterized by comprising the steps of sequentially soaking a porous ceramic support in solution A, solution C, solution B and solution D to obtain a double-layer multilayer film, repeating the steps at least once to obtain a plurality of double-layer multilayer films, soaking the double-layer multilayer films in an aqueous solution of a cross-linking agent for cross-linking for 30min to 24 hours, and fully cleaning the double-layer multilayer films in the washing solution to obtain the cross-linked composite ceramic support;

2. The preparation method according to claim 1, wherein the porous ceramic scaffold is a porous scaffold with interconnected pores, and the scaffold material comprises one or more of beta-TCP, hydroxyapatite and bioactive glass.

3. The method according to claim 1, wherein the solution A is an aqueous solution of one or more of chitosan, hyaluronic acid modified to have a positive charge, and polylysine.

4. The method according to claim 1, wherein the wash solution C and the wash solution D are both deionized water.

5. The method according to claim 1, wherein the solution B is an aqueous solution of one or more of hyaluronic acid, alginic acid, heparin, chondroitin sulfate and soluble salts thereof.

6. The preparation method according to claim 1, wherein the crosslinking agent is one or more of carbodiimide-based crosslinking agent, genipin, glutaraldehyde, and succinimide.

7. The method for preparing a composite ceramic scaffold according to claim 1, further comprising the step of soaking the crosslinked composite ceramic scaffold in a drug solution to load a drug that promotes bone repair.

8. The method of claim 7, wherein the drug solution is a solution of one or more of calcitriol, a bisphosphonate, strontium ranelate, dexamethasone.

9. The method of claim 8, wherein the solvent of the drug solution is ethanol.

10. A composite ceramic scaffold prepared by the method of any one of claims 1-9.