CN116036372B - Preparation method of bone filler for oral cavity - Google Patents
Preparation method of bone filler for oral cavity Download PDFInfo
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- CN116036372B CN116036372B CN202310207968.0A CN202310207968A CN116036372B CN 116036372 B CN116036372 B CN 116036372B CN 202310207968 A CN202310207968 A CN 202310207968A CN 116036372 B CN116036372 B CN 116036372B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/12—Phosphorus-containing materials, e.g. apatite
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Inorganic Chemistry (AREA)
- Materials For Medical Uses (AREA)
Abstract
The application relates to the technical field of bone fillers, in particular to a preparation method of an oral bone filler. The method comprises the following steps: (1) selection of raw materials: a. recombinant collagen; b. inorganic material: porous biphasic calcium phosphate ceramics; (2) dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to obtain a recombinant collagen solution; (3) preparing EDC solution and NHS solution; (4) Regulating the pH value of the recombinant collagen solution to be 5.5+/-2 by using a NaOH solution, slowly dropwise adding an EDC solution, and stirring while dropwise adding; then dripping NHS solution and stirring while dripping to obtain mixed solution; (5) Placing the mixed solution into a constant temperature shaking table for pre-crosslinking, and taking out to obtain a pre-crosslinked solution; (6) Weighing porous biphase calcium phosphate ceramic, adding into the pre-crosslinking solution, continuously placing into a constant temperature shaking table, crosslinking, taking out, and filtering out solid particles; (7) freeze-drying the solid particles to obtain the bone filler.
Description
Technical Field
The application relates to the technical field of bone fillers, in particular to a preparation method of an oral bone filler.
Background
Natural biological materials (animal collagen) have good biocompatibility, and more researchers apply animal collagen to the field of bone repair. However, animal collagen also has some problems, such as the potential virus hazards of mad cow disease and the like cannot be eliminated in the extraction process. In addition, human collagen cannot eliminate the infection risk of AIDS and the like in the extraction process. In addition, animal collagen has poor water solubility, and is easily cytotoxic in the course of dissolution in acids and bases. In summary, these problems with animal-and human-derived collagens have limited their use in the field of bone repair and the like. The recombinant collagen is a high molecular biological protein produced by a genetic engineering technology on the basis of human collagen genes, and has the advantages of good biocompatibility, easy water dissolution, low cytotoxicity, low rejection reaction, good cell adhesion and the like. Therefore, the recombinant collagen can be used as a substitute for animal collagen in conventional bone repair materials.
In the prior art, most of collagen bone repair materials are in a block shape, and have better mechanical strength, but poor plasticity, so that the collagen bone repair materials cannot be suitable for repairing various bone injury parts or irregular bone defect parts. In addition, most of collagen bone repair materials reported at present are prepared by a blending method and a coprecipitation method; the defects of easy agglomeration, uneven distribution, poor combination with a collagen interface and the like of inorganic materials (hydroxyapatite/beta-tricalcium phosphate) exist.
In summary, the above problems are solved, and an oral bone filler is prepared, which has better bioactivity and important value in different clinical application ways.
Disclosure of Invention
The application aims to provide a preparation method of an oral bone filler, which aims to solve the problems in the background technology.
In order to solve the technical problems, the application provides the following technical scheme:
a method for preparing bone filler for oral cavity, comprising the following steps:
(1) Selecting raw materials: a. recombinant collagen; b. inorganic material: porous biphasic calcium phosphate ceramics;
(2) Dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to obtain a recombinant collagen solution;
(3) Preparing a cross-linking agent: adding carbodiimide hydrochloride into distilled water, and dissolving to obtain EDC solution; adding N-hydroxysuccinimide into distilled water, and dissolving to obtain an NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5+/-2 by using a NaOH solution, slowly dropwise adding an EDC solution, and stirring while dropwise adding; then dripping NHS solution and stirring while dripping to obtain mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table for pre-crosslinking, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphase calcium phosphate ceramic, adding into the pre-crosslinking solution, continuously placing into a constant temperature shaking table, crosslinking, taking out, and filtering out solid particles;
(7) And freeze-drying the solid particles to obtain the bone filler.
More preferably, in the step (2), the concentration of the recombinant collagen solution is 80-300 mg/mL.
More preferably, in the step (3), the concentration of the EDC solution is 50-200 mg/mL; the concentration of the NHS solution is 20-100 mg/mL.
More preferably, in the step (4), the EDC solution accounts for 0.05 to 1 weight percent of the mixed solution; the NHS solution accounts for 0.01-1 wt% of the mixed solution.
More optimally, in the step (5), in the pre-crosslinking process, the temperature is adjusted to 30-40 ℃, the oscillation frequency is adjusted to 100-220 rpm, and the pre-crosslinking is performed for 30-50 minutes; in the step (6), in the crosslinking process, the temperature is adjusted to 30-40 ℃, the oscillation frequency is adjusted to 100-220 rpm, and the crosslinking time is 3-4 hours.
More preferably, in the step (6), the addition amount of the porous biphasic calcium phosphate ceramic accounts for 10-30wt% of the mixed solution.
More preferably, the granularity of the porous biphasic calcium phosphate ceramic is 0.25-1mm or 1-2 mm.
More optimally, the porous biphasic calcium phosphate ceramic consists of hydroxyapatite and beta-tricalcium phosphate in a mass ratio of 2:8.
More optimally, the bone filler prepared by the preparation method of the bone filler for the oral cavity is provided.
Compared with the prior art, the application has the following beneficial effects:
(1) The bone filler prepared in the application is a recombined gel type bone filler, has strong molding property, can be arbitrarily changed in shape, and is used for filling various irregular bone defect positions.
(2) According to the application, porous biphasic calcium phosphate ceramics (BCP particles: hydroxyapatite/tricalcium phosphate mixture) with different particle sizes are added into a recombined collagen solution pre-crosslinked by a crosslinking agent, so that the crosslinked recombined collagen is combined with the surface and the internal interface of the porous biphasic calcium phosphate ceramics, and the crosslinking distribution is uniform. The structural characteristics enable the compound to have better biological activity and different clinical application approaches.
(3) In the application, the concentration of the recombinant collagen solution and the granularity of the biphase calcium phosphate ceramic can be changed, so that the recombinant collagen type bone filler with different performance parameters is prepared, and is suitable for repairing various bone injury parts or irregular bone injury parts.
Drawings
The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate the application and together with the embodiments of the application, serve to explain the application. In the drawings:
FIG. 1 is an SEM image of the bone filler prepared in example 1;
FIG. 2 is an SEM image of the bone filler prepared in example 2;
FIG. 3 is an SEM image of the bone filler prepared in example 3;
FIG. 4 is an SEM image of the bone filler prepared in example 4;
FIG. 5 is an SEM image of the bone filler prepared in example 5;
FIG. 6 is an SEM image of the bone filler prepared in example 6;
FIG. 7 is an SEM image of the bone filler prepared in comparative example 1;
FIG. 8 is an SEM image of the bone filler prepared in comparative example 2;
FIG. 9 is an SEM image of the bone filler prepared in comparative example 3;
FIG. 10 is a macroscopic view of the bone filler prepared in example 6;
FIG. 11 is a macroscopic view of the bone filler prepared in comparative example 1;
FIG. 12 is a Micro-CT image of the implantation site of the bone powder of the group A rats;
FIG. 13 is a Micro-CT image of the bone meal implant site of group B rats;
FIG. 14 is a Micro-CT image of the bone meal implant site of group C rats;
FIG. 15 is a graph showing bone density results at the site of implantation of three groups of SD rat bone meal;
FIG. 16 is a graph of the volume percent of new bone at the site of implantation of three groups of SD rat bone meal;
FIG. 17 is a graph showing the ratio of bone surface area to tissue volume at the site of implantation of three groups of SD rat bone powder;
FIG. 18 is the in vitro degradation cumulative weight loss rate of bone meal of example 3;
figure 19 is the cumulative percent weight loss of bone meal in vitro degradation of example 6.
Detailed Description
The preferred embodiments of the present application will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present application only, and are not intended to limit the present application.
Example 1: (1) selection of raw materials: a. recombinant collagen: recombinant collagen type I (manufacturer: jiangsu Chuangjian medical science and technology Co., ltd., amino acid sequence and gene expression sequence of recombinant collagen are shown as SEQ ID NO.1 and SEQ ID NO.2 in patent number CN 110964099A); b. inorganic material: porous biphasic calcium phosphate ceramic (particle size 0.25-1mm, mass ratio of hydroxyapatite to beta-tricalcium phosphate 2:8);
(2) Dissolving collagen: adding the recombinant collagen into distilled water, stirring and dissolving to prepare 80mg/mL recombinant collagen solution;
(3) Preparing 100mg/mL EDC solution; preparing a 20mg/mL NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5 by using a NaOH solution with the concentration of 0.4mol/L, slowly dropwise adding an EDC solution with the concentration of 100mg/mL, stirring while dropwise adding, slowly dropwise adding an NHS solution with the concentration of 20mg/mL, and stirring while dropwise adding to obtain a mixed solution; wherein, EDC solution accounts for 0.2wt% of the mixed solution, NHS solution accounts for 0.2wt% of the mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 150rpm, pre-crosslinking for 30 minutes, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphasic calcium phosphate ceramic BCP to make the porous biphasic calcium phosphate ceramic BCP account for 20wt% of the mixed solution, adding the mixed solution into the pre-crosslinked solution, continuously placing the pre-crosslinked solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 150rpm, crosslinking for 3.5 hours, taking out, and filtering out solid particles;
(7) And freeze-drying the solid particles to obtain the bone filler.
Example 2: (1) selection of raw materials: a. recombinant collagen: recombinant collagen type I (manufacturer: jiangsu Chuangjian medical science and technology Co., ltd., amino acid sequence and gene expression sequence of recombinant collagen are shown as SEQ ID NO.1 and SEQ ID NO.2 in patent number CN 110964099A); b. inorganic material: porous biphasic calcium phosphate ceramic (particle size 0.25-1mm, mass ratio of hydroxyapatite to beta-tricalcium phosphate 2:8);
(2) Dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to prepare 170mg/mL recombinant collagen solution;
(3) Preparing 100mg/mL EDC solution; preparing a 20mg/mL NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5 by using a NaOH solution with the concentration of 0.4mol/L, slowly dropwise adding an EDC solution with the concentration of 100mg/mL, stirring while dropwise adding, slowly dropwise adding an NHS solution with the concentration of 20mg/mL, and stirring while dropwise adding to obtain a mixed solution; wherein, EDC solution accounts for 0.12wt% of the mixed solution, NHS solution accounts for 0.12wt% of the mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 130rpm, pre-crosslinking for 30 minutes, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphasic calcium phosphate ceramic BCP to make the porous biphasic calcium phosphate ceramic BCP account for 20wt% of the mixed solution, adding the mixed solution into the pre-crosslinked solution, continuously placing the pre-crosslinked solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 130rpm, crosslinking for 3.5 hours, taking out, and filtering out solid particles;
(7) And freeze-drying the solid particles to obtain the bone filler.
Example 3: (1) selection of raw materials: a. recombinant collagen: recombinant collagen type I (manufacturer: jiangsu Chuangjian medical science and technology Co., ltd., amino acid sequence and gene expression sequence of recombinant collagen are shown as SEQ ID NO.1 and SEQ ID NO.2 in patent number CN 110964099A); b. inorganic material: porous biphasic calcium phosphate ceramic (particle size 0.25-1mm, mass ratio of hydroxyapatite to beta-tricalcium phosphate 2:8);
(2) Dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to prepare 280mg/mL recombinant collagen solution;
(3) Preparing 100mg/mL EDC solution; preparing a 20mg/mL NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5 by using a NaOH solution with the concentration of 0.4mol/L, slowly dropwise adding an EDC solution with the concentration of 100mg/mL, stirring while dropwise adding, slowly dropwise adding an NHS solution with the concentration of 20mg/mL, and stirring while dropwise adding to obtain a mixed solution; wherein, EDC solution accounts for 0.1wt% of the mixed solution, NHS solution accounts for 0.1wt% of the mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 120rpm, pre-crosslinking for 30 minutes, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphasic calcium phosphate ceramic BCP to make the porous biphasic calcium phosphate ceramic BCP account for 20wt% of the mixed solution, adding the mixed solution into the pre-crosslinked solution, continuously placing the pre-crosslinked solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 120rpm, crosslinking for 3.5 hours, taking out, and filtering out solid particles;
(7) And freeze-drying the solid particles to obtain the bone filler.
Example 4: (1) selection of raw materials: a. recombinant collagen: recombinant collagen type I (manufacturer: jiangsu Chuangjian medical science and technology Co., ltd., amino acid sequence and gene expression sequence of recombinant collagen are shown as SEQ ID NO.1 and SEQ ID NO.2 in patent number CN 110964099A); b. inorganic material: porous biphasic calcium phosphate ceramic (particle size 1-2 mm, mass ratio of hydroxyapatite to beta-tricalcium phosphate 2:8);
(2) Dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to prepare 80mg/mL recombinant collagen solution;
(3) Preparing 100mg/mL EDC solution; preparing a 20mg/mL NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5 by using a NaOH solution with the concentration of 0.4mol/L, slowly dropwise adding an EDC solution with the concentration of 100mg/mL, stirring while dropwise adding, slowly dropwise adding an NHS solution with the concentration of 20mg/mL, and stirring while dropwise adding to obtain a mixed solution; wherein, EDC solution accounts for 0.2wt% of the mixed solution, NHS solution accounts for 0.2wt% of the mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 150rpm, pre-crosslinking for 30 minutes, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphasic calcium phosphate ceramic BCP to make the porous biphasic calcium phosphate ceramic BCP account for 20wt% of the mixed solution, adding the mixed solution into the pre-crosslinked solution, continuously placing the pre-crosslinked solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 150rpm, crosslinking for 3.5 hours, taking out, and filtering out solid particles;
(7) And freeze-drying the solid particles to obtain the bone filler.
Example 5: (1) selection of raw materials: a. recombinant collagen: recombinant collagen type I (manufacturer: jiangsu Chuangjian medical science and technology Co., ltd., amino acid sequence and gene expression sequence of recombinant collagen are shown as SEQ ID NO.1 and SEQ ID NO.2 in patent number CN 110964099A); b. inorganic material: porous biphasic calcium phosphate ceramic (particle size 1-2 mm, mass ratio of hydroxyapatite to beta-tricalcium phosphate 2:8);
(2) Dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to prepare 170mg/mL recombinant collagen solution;
(3) Preparing 100mg/mL EDC solution; preparing a 20mg/mL NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5 by using a NaOH solution with the concentration of 0.4mol/L, slowly dropwise adding an EDC solution with the concentration of 100mg/mL, stirring while dropwise adding, slowly dropwise adding an NHS solution with the concentration of 20mg/mL, and stirring while dropwise adding to obtain a mixed solution; wherein, EDC solution accounts for 0.12wt% of the mixed solution, NHS solution accounts for 0.12wt% of the mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 130rpm, pre-crosslinking for 30 minutes, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphasic calcium phosphate ceramic BCP to make the porous biphasic calcium phosphate ceramic BCP account for 20wt% of the mixed solution, adding the mixed solution into the pre-crosslinked solution, continuously placing the pre-crosslinked solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 130rpm, crosslinking for 3.5 hours, taking out, and filtering out solid particles;
(7) And freeze-drying the solid particles to obtain the bone filler.
Example 6: (1) selection of raw materials: a. recombinant collagen: recombinant collagen type I (manufacturer: jiangsu Chuangjian medical science and technology Co., ltd., amino acid sequence and gene expression sequence of recombinant collagen are shown as SEQ ID NO.1 and SEQ ID NO.2 in patent number CN 110964099A); b. inorganic material: porous biphasic calcium phosphate ceramic (particle size 1-2 mm, mass ratio of hydroxyapatite to beta-tricalcium phosphate 2:8);
(2) Dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to prepare 280mg/mL recombinant collagen solution;
(3) Preparing 100mg/mL EDC solution; preparing a 20mg/mL NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5 by using a NaOH solution with the concentration of 0.4mol/L, slowly dropwise adding an EDC solution with the concentration of 100mg/mL, stirring while dropwise adding, slowly dropwise adding an NHS solution with the concentration of 20mg/mL, and stirring while dropwise adding to obtain a mixed solution; wherein, EDC solution accounts for 0.1wt% of the mixed solution, NHS solution accounts for 0.1wt% of the mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 120rpm, pre-crosslinking for 30 minutes, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphasic calcium phosphate ceramic BCP to make the porous biphasic calcium phosphate ceramic BCP account for 20wt% of the mixed solution, adding the mixed solution into the pre-crosslinked solution, continuously placing the pre-crosslinked solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 120rpm, crosslinking for 3.5 hours, taking out, and filtering out solid particles;
(7) And freeze-drying the solid particles to obtain the bone filler.
Comparative example 1: (1) selection of raw materials: a. recombinant collagen: recombinant collagen type I (manufacturer: jiangsu Chuangjian medical science and technology Co., ltd., amino acid sequence and gene expression sequence of recombinant collagen are shown as SEQ ID NO.1 and SEQ ID NO.2 in patent number CN 110964099A); b. inorganic material: porous biphasic calcium phosphate ceramic (particle size 1-2 mm, mass ratio of hydroxyapatite to beta-tricalcium phosphate 2:8);
(2) Dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to prepare 280mg/mL recombinant collagen solution;
(3) Weighing biphasic calcium phosphate ceramic BCP to make the biphasic calcium phosphate ceramic BCP account for 20wt% of the recombinant collagen solution, preparing 100mg/mL EDC solution, and preparing 20mg/mL NHS solution;
(4) Adding the weighed biphasic calcium phosphate ceramic BCP into the recombinant collagen solution, placing the recombinant collagen solution into a constant-temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 120rpm, and premixing for 30min to obtain a premixed solution;
(5) Regulating the pH value of the premixed solution to 5.5 by using 0.4mol/L NaOH solution, slowly dropwise adding 100mg/ml EDC crosslinking agent solution, stirring while dropwise adding, slowly dropwise adding 20mg/ml NHS solution, and stirring while dropwise adding to obtain a mixed solution; wherein the EDC solution accounts for 0.1wt% of the total mixed solution, and the NHS solution accounts for 0.1wt% of the total mixed solution;
(6) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 120rpm, performing crosslinking reaction for 3.5 hours, and filtering out solid particles;
(7) And (3) freeze-drying the solid sample filtered in the step (6).
Comparative example 2: (1) selection of raw materials: a. recombinant collagen: recombinant collagen type I (manufacturer: jiangsu Chuangjian medical science and technology Co., ltd., amino acid sequence and gene expression sequence of recombinant collagen are shown as SEQ ID NO.1 and SEQ ID NO.2 in patent number CN 110964099A); b. inorganic material: porous biphasic calcium phosphate ceramic (particle size 1-2 mm, mass ratio of hydroxyapatite to beta-tricalcium phosphate 2:8);
(2) Dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to prepare 400mg/mL recombinant collagen solution;
(3) Preparing 100mg/mL EDC solution; preparing a 20mg/mL NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5 by using a NaOH solution with the concentration of 0.4mol/L, slowly dropwise adding an EDC solution with the concentration of 100mg/mL, and stirring while dropwise adding; slowly dropwise adding 20mg/mL of NHS solution, and stirring while dropwise adding to obtain a mixed solution; wherein, EDC solution accounts for 0.1wt% of the mixed solution, NHS solution accounts for 0.1wt% of the mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillation frequency to 120pm, pre-crosslinking for 30 minutes, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphasic calcium phosphate ceramic BCP to make the porous biphasic calcium phosphate ceramic BCP account for 20wt% of the mixed solution, adding the mixed solution into the pre-crosslinked solution, continuously placing the pre-crosslinked solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 120rpm, crosslinking for 3.5 hours, taking out, and filtering out solid particles;
(7) And freeze-drying the solid particles to obtain the bone filler.
Comparative example 3: (1) selection of raw materials: a. recombinant collagen: recombinant collagen type I (manufacturer: jiangsu Chuangjian medical science and technology Co., ltd., amino acid sequence and gene expression sequence of recombinant collagen are shown as SEQ ID NO.1 and SEQ ID NO.2 in patent number CN 110964099A); b. inorganic material: porous biphasic calcium phosphate ceramic (particle size 1-2 mm, mass ratio of hydroxyapatite to beta-tricalcium phosphate 2:8);
(2) Dissolving collagen: adding the recombinant collagen into distilled water, dissolving, and preparing 40mg/mL recombinant collagen solution;
(3) Preparing 100mg/mL EDC solution; preparing a 20mg/mL NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5 by using a NaOH solution with the concentration of 0.4mol/L, slowly dropwise adding an EDC solution with the concentration of 100mg/mL, and stirring while dropwise adding; slowly dropwise adding 20mg/mL of NHS solution, and stirring while dropwise adding to obtain a mixed solution; wherein, EDC solution accounts for 0.1wt% of the mixed solution, NHS solution accounts for 0.1wt% of the mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 120rpm, pre-crosslinking for 30 minutes, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphasic calcium phosphate ceramic BCP to make the porous biphasic calcium phosphate ceramic BCP account for 20wt% of the mixed solution, adding the mixed solution into the pre-crosslinked solution, continuously placing the pre-crosslinked solution into a constant temperature shaking table, adjusting the temperature to 37 ℃, adjusting the oscillating frequency to 120rpm, crosslinking for 3.5 hours, taking out, and filtering out solid particles;
(7) And freeze-drying the solid particles to obtain the bone filler.
Experimental part:
experiment 1: the bone fillers prepared in examples and comparative examples were subjected to collagen content measurement and SEM scanning electron microscopy, and at the same time, the products obtained in example 6 and comparative example 1 were subjected to physical photographing, and the data obtained are shown in the following table:
conclusion: the SEM scanning electron microscope charts in examples 1 to 6 show that: collagen is distributed in a strip shape between porous biphasic calcium phosphate ceramic BCPs.
Comparative example 1 was compared to example 6, wherein the process steps and parameters of comparative example 1 and example 6 were substantially the same, except that example 6 was a cross-linking of the reconstituted collagen solution followed by immersing BCP particles in the cross-linking solution, i.e. cross-linking followed by immersing; in comparative example 1, the BCP particles were soaked in the recombinant collagen solution, premixed for 30min, and then crosslinked with the recombinant collagen solution, i.e., soaked before crosslinked. From fig. 10 and 11, it can be seen that: example 6 the bone filler obtained by the cross-linking followed by the soaking process was in the form of granules, whereas the bone filler obtained by the cross-linking followed by the soaking process of comparative example 1 was in the form of blocks. As can be seen by comparing fig. 6 and 7 again: in the bone filler of example 6, collagen is distributed among BCP particles in a stripe shape, so that BCP is prevented from being completely wrapped, and the obtained bone filler is in a small particle shape, has strong plasticity, and can be arbitrarily changed in shape to fill various irregular bone defect positions. In contrast, in the bone filler of comparative example 1, collagen completely encapsulates BCP particles in a film form, and separation is difficult.
Comparative example 2 was compared with example 6, wherein the processes of comparative example 2 and example 6 were substantially the same, except that the concentration of the recombinant collagen solution initially configured in comparative example 2 was higher. Thus, the collagen content of the bone filler of comparative example 2 was higher than that of example 6. And comparing fig. 6 and 8, it can be seen that: in the bone filler of comparative example 2, collagen completely encapsulates BCP particles in a film form, and thus cannot be separated, and thus is difficult to apply to various irregular bone defect sites.
Comparative example 3 was compared with example 6, wherein the processes of comparative example 3 and example 6 were substantially the same, except that the concentration of the recombinant collagen solution initially prepared in comparative example 3 was lower. Thus, the collagen content of the bone filler of comparative example 3 is significantly lower than that of example 6. And comparing fig. 6 with fig. 9, it can be seen that: in the bone filler of comparative example 3, no significant collagen was found to be present.
Experiment 2: the bone filler prepared in example 3 was subjected to animal experiments.
a. Preparation of animals prior to experiments:
(1) 15 adult male SD rats were randomly divided into 3 groups (group A, group B, group C), each group being fed 5 separately;
(2) Material preparation: the bone powder prepared in example 3 was sterilized by ethylene oxide and a Biooss bone powder (0.25-1 mm particle size) of Gauss was prepared as a control.
(3) Surgical instrument preparation: the necessary surgical instruments are wrapped by cloth and put into a metal container, and a high-pressure steam sterilizing pot is used for sterilizing.
(4) Preparation of the gunpowder: the weighed 2g sodium pentobarbital powder is added into a small amount of physiological saline to be fully dissolved, and is filtered into a sterile solution by a sterile filter tip and then is injected into 100mL of physiological saline to prepare a 2% sodium pentobarbital-physiological saline solution.
Establishing a SD rat skull defect model:
the SD rat is anesthetized by injecting 1mL of 2% pentobarbital sodium-physiological saline solution into the abdominal cavity of the SD rat by a 1mL syringe, the hair at the top of the head of the SD rat is shaved by an electric shaver after the anesthesia, the iodophor is sterilized for 2 times, a 1.5cm incision is made on the median line of Fang Lugu on the skull of the rat, the epidermis, the subcutaneous fascia and the periosteum are sequentially cut, a skull herringbone seam can be seen, and the incision can be properly prolonged if insufficient exposure is achieved. The punctiform scars are uniformly made by a mould with the diameter of 6cm beside the herringbone joint, and the equal-sized full-layer bone defects are manufactured along the scars, so that people do not hurt the brain, and the bleeding is stopped by pressing in time so as not to influence the repairing effect. After the bone defect is produced, as shown in the following table, the corresponding materials (group A is not implanted with bone powder, group B is implanted with bone powder prepared in example 3, group C is implanted with Gauss Bio-oss bone powder) are implanted in the corresponding groups, the skin is directly sutured under the skin after operation, 5mL of glucose is injected, and the bone is kept warm under an incandescent lamp for half an hour, so that the bone defect can be put back into a feeder cage. The experimental rats are injected with glucose solution and penicillin solution daily for 3 days before operation to resist inflammation, the infection phenomenon of the operation wound is observed and treated in time, and the materials are observed and obtained 2 months after operation to carry out relevant detection.
c. Results processing (detection):
2 months after the SD rat skull defect model is subjected to material repair, an animal specimen Micro-CT scan is performed. Two groups of SD rats were anesthetized with 2% sodium pentobarbital, sacrificed by cervical dislocation, the rat cranium was completely removed, and after 1 week of soaking with formaldehyde, micro-CT scan was performed.
d. Results and discussion
As shown in FIGS. 12 to 14, the results of the Micro-CT scan are respectively shown as Micro-CT images of rats of group A (bone powder-free implantation), group B (bone powder implantation of example 3) and group C (Bio-oss bone powder implantation) in this order, and it can be seen that: group a (bone-free implant) rats had minimal new bone formation, and group B (implant with bone-powder of example 3) rats had more and more dense new bone formation than group C (implant with Bio-oss-powder).
While figures 15-17 show the bone density results, the volume percent of new bone, the ratio of bone surface area to tissue volume for three groups of SD rat bone powder implantation sites, the data can be seen: group B rats implanted with the bone powder of example 3 were excellent in terms of new bone formation, both in terms of bone density and volume percent of new bone, over group a (no bone powder implant) and group C (Bio-oss bone powder implant) rats.
Experiment 3: in vitro degradation experiments are carried out on the bone meal samples prepared in the embodiment 3 and the embodiment 6, the prepared bone meal samples are placed in a simulated body fluid SBF solution with the pH value of 7, the bone meal samples are shaken at a low speed in a constant temperature shaking table with the temperature of 37 ℃, taken out every 7d, placed in an oven, dried and weighed at the temperature of 40 ℃, and the bone meal cumulative weight loss rate is calculated.
Conclusion: the degradation experimental data of example 3 is shown in fig. 18, which shows that: the bone powder prepared in example 3 has a cumulative weight loss rate of less than 15% after in vitro degradation for 3 months, and a slow degradation rate, and meets the degradation requirements of the bone filling material for oral cavity.
The degradation experimental data of example 6 is shown in fig. 19, which shows that: the data indicate that: after the bone powder prepared in the example 6 is degraded in vitro for 3 months, the accumulated weight loss rate is less than 10%, the degradation rate is slower, and the degradation requirement of the bone filling material for oral cavity is met.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present application, and the present application is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present application has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (6)
1. A method for preparing bone filler for oral cavity, which is characterized in that: the method comprises the following steps:
(1) Selecting raw materials: a. recombinant collagen; b. inorganic material: porous biphasic calcium phosphate ceramics;
(2) Dissolving collagen: adding the recombinant collagen into distilled water, and dissolving to obtain a recombinant collagen solution with the concentration of 80-300 mg/mL;
(3) Preparing a cross-linking agent: adding carbodiimide hydrochloride into distilled water, and dissolving to obtain EDC solution; adding N-hydroxysuccinimide into distilled water, and dissolving to obtain an NHS solution;
(4) Regulating the pH value of the recombinant collagen solution to be 5.5+/-2 by using a NaOH solution, slowly dropwise adding an EDC solution, and stirring while dropwise adding; then dripping NHS solution and stirring while dripping to obtain mixed solution;
(5) Placing the mixed solution into a constant temperature shaking table, adjusting the temperature to 30-40 ℃, adjusting the oscillation frequency to 100-220 rpm, pre-crosslinking for 30-50 minutes, and taking out to obtain a pre-crosslinked solution;
(6) Weighing porous biphase calcium phosphate ceramic, adding the porous biphase calcium phosphate ceramic into a pre-crosslinking solution, continuously placing the solution into a constant temperature shaking table, adjusting the temperature to 30-40 ℃, adjusting the oscillating frequency to 100-220 rpm, crosslinking for 3-4 hours, taking out, and filtering out solid particles; the addition amount of the porous biphase calcium phosphate ceramic accounts for 10-30wt% of the mixed solution;
(7) And freeze-drying the solid particles to obtain the bone filler.
2. The method for preparing the bone filler for oral cavity according to claim 1, wherein: in the step (3), the concentration of the EDC solution is 50-200 mg/mL; the concentration of the NHS solution is 20-100 mg/mL.
3. The method for preparing the bone filler for oral cavity according to claim 1, wherein: in the step (4), the EDC solution accounts for 0.05 to 1 weight percent of the mixed solution; the NHS solution accounts for 0.01-1 wt% of the mixed solution.
4. The method for preparing the bone filler for oral cavity according to claim 1, wherein: the granularity of the porous biphasic calcium phosphate ceramic is 0.25-1mm or 1-2 mm.
5. The method for preparing the bone filler for oral cavity according to claim 1, wherein: the porous biphasic calcium phosphate ceramic consists of hydroxyapatite and beta-tricalcium phosphate in a mass ratio of 2:8.
6. The bone filler according to any one of claims 1 to 5, which is prepared by a method for preparing a bone filler for oral cavity.
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| JP7476240B2 (en) * | 2019-06-14 | 2024-04-30 | ガイストリッヒ ファーマ アーゲー | Collagen matrix or granular blend of bone substitute |
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