CN1338315A - Process for preparing nm-crysal collagen-based calcium phosphate composition used for repairing bone - Google Patents

Abstract

The invention relates to a method for preparing a nanocrystalline calcium phosphate collagen-based composite material for bone repair. First, a solution containing PO ₄ ^3- is added dropwise to an acid-soluble collagen solution, and a solution containing Ca ² is slowly added dropwise to the solution. ⁺ aqueous solution, and finally NaOH solution was added dropwise, the above solution was left to stand to remove the supernatant, the precipitate was separated by centrifugation, after washing, it was freeze-dried, and ground into a dry powder, which was the bone repair material. The bone repair material prepared by the method of the present invention is similar to natural bone in terms of inorganic and organic components, and the crystal size of the calcium phosphate salt is in the nanometer order, and is expected to be used for preparing bone substitute materials with excellent biological properties.

Description

Preparation method of nanocrystalline calcium phosphate collagen-based composite material for bone repair

Technical field:

The invention relates to a method for preparing a nanocrystalline calcium phosphate collagen-based composite material for bone repair, belonging to the technical field of bioengineering.

The research and development of bone repair materials has always been the direction of a large number of scientists in the field of biomedical engineering. The best bone repair materials actually used in surgery are autologous bone and allogeneic bone. However, the amount of autologous bone is limited, while allogeneic bone has the risk of immune rejection and infection. People have developed a variety of bone repair materials made of metals, ceramics, polymers and their composite materials. However, these materials have their own deficiencies, none of which can be compared with autologous bone or allogeneic bone in clinical practice. Considering that the biological advantages of autologous bone and allogeneic bone lie in that their composition and structure are exactly the same as those of natural bone, the principle of bone tissue engineering holds that a material with similar composition and microstructure to natural bone will have excellent biological properties. performance.

Natural bone is a complex biomineralization system with a finely graded structure. Collagen and calcium phosphate are the main substances present in bone. Calcium-phosphorus salts in bone are mainly irregularly shaped sheet-like nanocrystals with roughly parallel c-axes. The crystal width is about 30-45nm and the thickness is 4-6nm. Adjacent lamellar crystals constitute calcium-phosphorus salt crystal layers, which are alternately arranged with collagen layers in space, and the lamellar spacing is on the order of nanometers, and the period is about 10nm. In addition to calcium phosphate crystals, highly ordered collagen acts as an extracellular matrix and has an important influence on cell activity.

Invention content:

The purpose of the present invention is to propose a method for preparing a nanocrystalline calcium phosphate collagen-based composite material for bone repair, to prepare a nanocrystalline calcium phosphate collagen-based composite material self-assembled from nanophase calcium phosphate and collagen molecules. The product of the material has a repeating lamellar structure on the nanometer scale, with a period of 10-15nm, and is formed by alternate arrangements of collagen layers and calcium phosphate salt layers. This material imitates natural bone in composition and structure, so the obtained material will have good biocompatibility and high bioactivity. Combining this material with other frame materials can be used to prepare bone repair materials with excellent biological and mechanical properties.

A method for preparing a nanocrystalline calcium phosphate collagen-based composite material for bone repair proposed by the present invention is characterized in that the method comprises the following steps:

(1) Slowly add a solution containing PO ₄ ^3- into the acid-soluble collagen solution so that each gram of collagen contains 0.01 to 0.1 mol PO ₄ ^3- , and stir while adding. The acid here can be hydrochloric acid, nitric acid, Acetic acid, etc., the concentration of collagen solution is 5.0×10 ^-5 ~5.0×10 ^-3 g/ml;

(2) Slowly add the aqueous solution containing Ca ²⁺ dropwise while stirring in the solution prepared in the above-mentioned first step, the mol ratio of the added Ca ²⁺ amount to the P added in the first step is Ca: P=1～2 : 1;

(3) Slowly add NaOH solution dropwise while stirring in the solution of the second step above, so that the pH value is 6-8, and precipitation begins to appear when the pH value is 5-6, and a white suspension appears when the pH value is 7 ;

(4) Let the above solution stand for 1 to 5 days, remove the supernatant, and centrifuge to separate the precipitate. After repeated washing with deionized water three times, put it into a freeze dryer to freeze dry, and then grind the obtained dry powder, which is bone bone powder. Restoration materials.

The nanocrystalline calcium phosphate collagen-based composite material for bone repair prepared by the method of the invention is not only similar to natural bone in inorganic and organic components, but also has a certain degree of bone imitation in structure. The crystal size of the calcium phosphate salt is at the nanometer level, closely combined with the organic component collagen, and arranged in a certain order. Its biocompatibility and bioactivity are very good and can be used as bone filling materials, and it is expected to be used to prepare bone substitute materials with excellent biological properties when combined with other framework materials.

Collagen used in the present invention is type I collagen gel (concentration: 1% of solid content, contained collagen is purified cowhide collagen) of Yierkang Company, liquid type I collagen (concentration: 0.3% solution pH value) purchased by CELLON company The collagen contained in it was adjusted by HCl to be purified cowhide collagen), type I collagen gel from the Second Affiliated Hospital of Tianjin (concentration: solid content 1%, contained collagen was purified bovine Achilles tendon collagen); the used PO ₄ ³ The solution of ^- is Na2HPO ₄ , H ₃ PO ₄ or (NH ₄ ) ₂ HPO ₄ aqueous solution; the solution containing Ca ²⁺ used is CaCl ₂ 6H ₂ O, Ca(NO ₃ ) ₂ or CaCl ₂ 2H ₂ Aqueous solution of O.

Detailed ways:

Introduce the embodiment of the present invention below:

Example 1:

The materials used are type I collagen gel (concentration: 1% solid content, the collagen contained is purified cowhide collagen), analytically pure CaCl ₂ ·6H ₂ O, and analytically pure Na ₂ HPO ₄ purchased from Yierkang Company.

1) Dissolve 20g collagen gel in 300ml 0.5M acetic acid solution, slowly add 11ml 1mol/l Na ₂ HPO ₄ and 18.3 ml 1mol/l CaCl ₂ dropwise, and stir with a magnetic stirrer while adding dropwise;

2) Continue stirring while slowly adding 0.5mol/l NaOH solution dropwise to a pH value of 7;

3) Let the solution stand for 1 day, remove the supernatant, centrifuge to separate the precipitate, wash it three times with deionized water, put it into a freeze dryer for freeze drying, and grind it to obtain a dry powder.

Example 2:

The materials used are liquid type I collagen purchased by CELLON company (concentration: the pH value of the 0.3% solution is adjusted by HCl and the contained collagen is purified cowhide collagen), analytically pure CaCl ₂ 6H ₂ O, analytically pure H ₃ PO ₄ (content >=85% density 1.689 g/ml).

(1) Add dropwise 1.125ml H ₃ PO ₄ dissolved in 10ml deionized water to 100ml collagen solution, and stir with a magnetic stirrer while adding dropwise;

(2) Weigh 6.01 g of well-crystallized CaCl ₂ 6H ₂ O and dissolve it in 20 ml of deionized water to dissolve it completely, add it dropwise to the solution prepared in the previous step, and continue stirring for 1 hour;

(3) continue to stir, slowly dropwise simultaneously the NaOH solution of 0.75mol/l to pH value 7;

(4) Let the solution stand for 1 day, remove the supernatant, centrifuge to separate the precipitate, wash it repeatedly with deionized water three times, put it into a lyophilizer for lyophilization, and grind it to obtain a dry powder.

Example 3:

The materials used are type I collagen gel (concentration: 1% solid content, the collagen contained is purified cowhide collagen), analytically pure Ca(NO ₃ ) ₂ , and analytically pure Na ₂ HPO ₄ purchased from Yierkang Company.

1) Dissolve 20g collagen gel in 300ml 0.5M acetic acid solution, slowly add 11ml 1mol/l H ₃ PO ₄ and 16.5 ml 1mol/l Ca(NO ₃ ) ₂ dropwise, and stir with a magnetic stirrer while adding dropwise;

2) Continue stirring while slowly adding 0.5mol/l NaOH solution dropwise to a pH value of 7;

3) Let the solution stand for 1 day, remove the supernatant, centrifuge to separate the precipitate, wash it three times with deionized water, put it into a freeze dryer for freeze drying, and grind it to obtain a dry powder.

Example 4:

The materials used are type I collagen gel from Tianjin Second Affiliated Hospital (concentration: solid content 1%, the collagen contained is purified bovine Achilles tendon collagen), analytically pure CaCl ₂ ·2H ₂ O, analytically pure (NH ₄ ) _{2 HPO4} .

1) Dissolve 8g collagen gel in 300ml 0.1M HNO ₃ solution, slowly add 10ml 1mol/l (NH ₄ ) ₂ HPO ₄ and 10ml 1mol/l CaCl ₂ dropwise, and stir with a magnetic stirrer while adding dropwise;

2) Continue stirring while slowly adding 0.5mol/l NaOH solution dropwise to a pH value of 7;

3) Let the solution stand for 4 days, remove the supernatant, centrifuge to separate the precipitate, wash it repeatedly with deionized water three times, put it into a lyophilizer for lyophilization, and grind it to obtain a dry powder.

Example 5:

The materials used are type I collagen gel (concentration: 1% solid content, the collagen contained is purified cowhide collagen), analytically pure CaCl ₂ ·6H ₂ O, and analytically pure Na ₂ HPO ₄ purchased by Yierkang Company.

1) Dissolve 30g collagen gel in 300ml 0.5M acetic acid solution, slowly add 11ml 1mol/l Na2HPO ₄ and 18.3ml 1mol/l CaCl ₂ dropwise, and stir with a magnetic stirrer while adding dropwise;

2) Continue stirring while slowly adding 0.5mol/l NaOH solution dropwise to a pH value of 7;

3) Let the solution stand for 5 days, remove the supernatant, centrifuge to separate the precipitate, wash it three times with deionized water, put it into a freeze dryer for freeze drying, and grind it to obtain a dry powder.

Claims (1)

1. A method for preparing a nanocrystalline calcium phosphate collagen-based composite material for bone repair, characterized in that the method comprises the following steps: (1) Slowly add a solution containing PO ₄ ^3- into the acid-soluble collagen solution, so that each gram of collagen contains 0.01-0.1mol PO ₄ ^3- , stir while adding, and the concentration of the collagen solution is 5.0×10 ^-5 ～5.0×10 ^-3 g/ml; (2) Slowly add the aqueous solution containing Ca ²⁺ dropwise while stirring in the solution prepared in the above-mentioned first step, the mol ratio of the added Ca ²⁺ amount to the P added in the first step is Ca: P=1～2 : 1; (3) Slowly add NaOH solution dropwise while stirring in the solution of the second step above, so that the pH value is 6 to 8; (4) Let the above solution stand for 1 to 5 days, remove the supernatant, and centrifuge to separate the precipitate. After repeated washing with deionized water three times, put it into a freeze dryer to freeze dry, and then grind the obtained dry powder, which is bone bone powder. Restoration materials.