CN105148324A - Bone bionic materials built by mineralized nano cellulose whiskers and production method of bone bionic materials - Google Patents

Abstract

The invention relates to a bone bionic material constructed from mineralized nano-cellulose whiskers and a preparation method thereof. It grafts a large number of carboxyl groups on the surface of nano-cellulose whiskers through the action of sodium hydroxide and sodium chloroacetate. Under strong stirring, Ca(OH) ₂ suspension and H ₃ PO ₄ are added dropwise at the same time according to the molar ratio of calcium to phosphorus of 1.67. Put the solution into the nanocellulose suspension solution, control the pH value of the solution to 7.0-11.0, and obtain nanocellulose whiskers/hydroxyapatite composite particles, add the nanocomposite particles to silk fibroin solutions of different concentrations to fully reconcile and evenly Put it into a self-made mold, and freeze-dry layer by layer according to the order of mass concentration from high to low to prepare nano-bone biomimetic materials. The bone biomimetic material constructed by mineralized nano-cellulose whiskers prepared by the present invention can biomime natural bone in terms of composition and structure, and has good mechanical properties, reasonable porosity, good biocompatibility and bioactivity , can be used as a repair or replacement material for bone tissue, and has broad application prospects.

Description

A bone biomimetic material constructed from mineralized nanocellulose whiskers and its preparation method

technical field

The invention relates to a bone biomimetic material constructed from mineralized nano-cellulose whiskers and a preparation method thereof, which is applied to bone repair or bone replacement materials, and specifically relates to the technical field of biomedical materials.

Background technique

In the field of orthopedics, bone defects caused by trauma, infection, bone disease, bone tumor resection, etc. are very common. Both autologous bone and allogeneic bone have problems such as increased surgical complications, limited material sources, disease transmission or immune rejection, and their clinical application has been limited to a certain extent. With the development of biomaterial technology, artificial bone substitute materials are increasingly used clinically. Bone substitute material is mainly used as a framework placed in human bone, on which bone cells and collagen fibers grow, and finally become a part of living bone. Therefore, finding an ideal bone graft substitute material has been the goal of people's efforts.

At present, there are several categories of metals, ceramics and polymers used for bone tissue repair. However, metal materials lack the biological activity to combine with human tissue. Ceramic materials mainly have shortcomings such as brittleness, easy fracture in the body, and fatigue damage. Polymers can have a modulus similar to human bone, but they do not have the ability to form biological bonds with bone tissue. activity. Therefore, finding an ideal material for bone tissue repair is still a clinical difficulty. Natural bone is an organic/inorganic biocomposite material composed of about 30% organic matrix and 70% hydroxyapatite. The organic matrix mainly includes non-collagen, mucopolysaccharide, etc. Hydroxyapatite nanocrystals are orderly embedded in the collagen fiber matrix. The close synergy between organic and inorganic components and the unique assembly at the molecular level endow natural bone with a multi-level structure and excellent mechanical properties. Therefore, in order to prepare bone repair or bone replacement materials with good biocompatibility and mechanical compatibility and bioactivity, it is necessary to design biomimetic design at the molecular level to mimic the structure and function of natural bone. Nanowhiskers are a kind of nanoscale cellulose extracted from natural fibers. It not only has the characteristics of nanoparticles, but also has some unique strength and optical properties, and has broad application prospects. Through carboxylation modification, a large number of -COOH groups are grafted on the surface of cellulose nano whiskers, which aims to strengthen the functionality of nano whiskers without breaking the inherent advantages, and expands its application range.

Contents of the invention

The purpose of the present invention is to provide a preparation method of a bone biomimetic material constructed from mineralized nano-cellulose whiskers. Nano-cellulose whiskers/hydroxyapatite nanocomposite particles were obtained by carboxylation modification of nano-cellulose whiskers and biomimetic mineralization by electrostatic self-assembly, which were blended with different concentrations of silk fibroin to obtain density and Porous bone biomimetic material with gradient distribution of porosity. This bone biomimetic material not only simulates natural bone in composition, but also mimics natural bone in structure, and has good biocompatibility, biodegradability, osteoinductive properties, and excellent mechanical properties.

The technical solution for realizing the object of the present invention is a bone biomimetic material constructed by mineralized nano-cellulose whiskers, which is composed of inorganic carboxylated nano-cellulose whiskers, hydroxyapatite and organic silk fibroin. The mass ratio of the inorganic component carboxymethylated nano-whiskers to hydroxyapatite is 1:0-1, and the mass ratio of the inorganic component to the organic component is 1:0.5-2.

The bone biomimetic material is composed of carboxylated nano-cellulose whiskers, hydroxyapatite and silk fibroin, and the mass ratio of carboxylated nano-cellulose whiskers, hydroxyapatite and silk fibroin is 1:1-10: 0.5-5, the density gradient from outside to inside is 75.3%-82.3%, 57.4%-67.2%, 11.6%-30.6%.

The aspect ratio of the nano whiskers is about 20:1-2, the silk fibroin is mulberry silk fibroin and tussah silk fibroin, the intrinsic viscosity of the protein molecule is greater than or equal to 0.50, and the hydroxyapatite Stone presents acicular distribution, the particle length is 50-300nm, and the particle width is 10-50nm.

The method for preparing this bone biomimetic material constructed from mineralized nanocellulose whiskers adopts the following steps:

(1) Ultrasonically disperse nanocellulose whiskers into ethanol solution of 0.1-2mol/L sodium hydroxide with a liquor ratio of 1:500, and alkalize at 20-80°C for 10-120min. Then add 0.125-2.5mol/L sodium chloroacetate solution, ultrasonically stir at 30-75°C for 15-100min, adjust the pH value of the solution to neutral with 0.1-0.5mol/L hydrochloric acid and dialyze in distilled water for three days, Obtain carboxylated modified nano-cellulose crystal suspension solution;

(2) According to the ratio of calcium and phosphorus substances being 1.67, drop 0.01-0.1mol/L Ca(OH) ₂ and diluted H ₃ PO ₄ solution into the solution prepared in step (1) simultaneously with a burette and Stir in ultrasonic for 1-6h, add ammonia water dropwise to control the reaction pH to 8.0-10.0, control the reaction temperature to 20-80°C, and obtain a composite with a ratio of nanocellulose whiskers/hydroxyapatite of 1:1-10 Particle suspension;

(3) Leave the suspension of nanocellulose whiskers/hydroxyapatite nanocomposite particles obtained in step (2) at room temperature for 48 hours, remove the supernatant, centrifuge to separate the precipitate, wash with distilled water three times, and freeze-dry Prepare nano-cellulose whiskers/hydroxyapatite composite particles;

(4) Ultrasonically disperse the nanocellulose whiskers/hydroxyapatite composite particles obtained in step (3) into silk fibroin solutions with concentrations of 5%-15%, 35%-45%, and 55%-65% respectively , obtaining different concentrations of nanocellulose whiskers/hydroxyapatite/silk fibroin blend solutions, wherein the mass ratio of nanocellulose whiskers/hydroxyapatite nanocomposite particles to silk fibroin is 65:35;

(5) Pour the nanocellulose whisker/hydroxyapatite/silk fibroin blend solution with the highest mass concentration prepared in step (4) into a self-made mold, quench in liquid nitrogen, and store at -50 --Freeze-dry at 80°C, and then freeze-dry other blended solutions layer by layer according to the order of mass concentration from high to low to obtain a nano-particle containing graphene, hydroxyapatite and silk fibroin with a gradient distribution of density and porosity. Bone biomimetic materials.

Compared with the existing bone substitute material and its preparation method, the present invention has the following advantages:

(1) In the composite particles self-assembled from carboxylated nanocellulose whiskers and hydroxyapatite in the present invention, rod-shaped hydroxyapatite is distributed on the surface of nanocellulose whiskers due to electrostatic interaction, The rod-shaped nanocellulose whiskers are connected together, and the carboxylated nanocellulose whiskers and hydroxyapatite layers are alternately arranged to form a steel-concrete structure. Therefore, the compressive strength and toughness of hydroxyapatite can be improved;

(2) The porous bone biomimetic material prepared in the present invention has a pore structure with gradient distribution, so that it can biomime natural bone macroscopically. The composite scaffold material prepared by the invention can easily obtain the desired shape and size; can form pores with a certain structure and content, and can meet the requirements for bone bionic materials in the process of bone transplantation;

(3) The bone biomimetic material prepared in the present invention has non-toxicity, non-irritation, good biocompatibility, good cell affinity, good biodegradability and excellent mechanical properties.

The bone biomimetic material constructed by mineralized nano-cellulose whiskers prepared by the present invention is mainly based on carboxymethylated modified nano-cellulose whiskers, and obtains nano-cellulose whiskers/hydroxyl groups through electrostatic self-assembly biomimetic mineralization Apatite composite particles combined with silk fibroin to prepare porous bone biomimetic materials with gradient distribution of density and porosity.

Description of drawings

The morphology of nanocellulose whiskers after Fig. 1 carboxymethylation;

Fig. 2 is the morphology of the composite particles whose ratio of cellulose nano whiskers and hydroxyapatite is 1:1;

Fig. 3 cellulose nano whiskers and hydroxyapatite ratio are the appearance of the composite particle of 1:2;

Fig. 4 Morphology of composite particles with a ratio of cellulose nanowhiskers to hydroxyapatite of 1:4.

Detailed ways

The present invention will be further described in detail below by examples in conjunction with the accompanying drawings.

A bone biomimetic material constructed from mineralized nano-cellulose whiskers, which is composed of inorganic carboxylated nano-cellulose whiskers and hydroxyapatite and organic silk fibroin, the inorganic component carboxymethylated nano The mass ratio of whiskers to hydroxyapatite is 1:0-1, the mass ratio of the inorganic components to organic components is 1:0.5-2, and the bone biomimetic material is composed of carboxylated nano-cellulose whiskers, Composed of hydroxyapatite and silk fibroin, the mass ratio of carboxylated nanocellulose whiskers, hydroxyapatite and silk fibroin is 1:1-10:0.5-5, and the density gradient from outside to inside is 75.3 %-82.3%, 57.4%-67.2%, 11.6%-30.6%. The aspect ratio of the nano whiskers is about 20:1-2, the silk fibroin is mulberry silk fibroin and tussah silk fibroin, the intrinsic viscosity of the protein molecule is greater than or equal to 0.50, and the hydroxyapatite Stone presents acicular distribution, the particle length is 50-300nm, and the particle width is 10-50nm.

Example 1

A preparation method of a bone biomimetic material constructed by mineralized nano-cellulose whiskers is carried out according to the following steps:

(1) Ultrasonic disperse nanocellulose whiskers into 0.5mol/L sodium hydroxide ethanol (85%) solution with a bath ratio of 1:500, alkalinize at 30°C for 60min, then add 0.725mol/L Sodium chloroacetate solution was ultrasonically stirred at 60°C for 50 minutes, and the pH value of the solution was adjusted to neutral with 0.1mol/L hydrochloric acid, and dialyzed in distilled water for three days to obtain a suspension solution of carboxylated modified nanocellulose whiskers;

(2) According to the ratio of the amount of calcium and phosphorus substances being 1.67, drop 0.05mol/L Ca(OH) ₂ and diluted H ₃ PO ₄ solution into the solution prepared in step (1) simultaneously with a burette and ultrasonically Stir in medium for 4 hours, add ammonia water dropwise to control the reaction pH to 8.0-9.0, and control the reaction temperature to 60°C to obtain a composite particle suspension with a nanocellulose whisker/hydroxyapatite ratio of 1:1;

(3) Leave the suspension of nanocellulose whiskers/hydroxyapatite nanocomposite particles obtained in step (2) at room temperature for 48 hours, remove the supernatant, centrifuge to separate the precipitate, wash with distilled water three times, and freeze-dry Prepare nano-cellulose whiskers/hydroxyapatite composite particles;

(4) Ultrasonic disperse nanocellulose whiskers/hydroxyapatite composite particles obtained in step (3) into silk fibroin solutions with concentrations of 5%, 35, and 55% respectively to obtain nanocellulose whiskers with different concentrations /hydroxyapatite/silk fibroin blend solution, wherein the mass ratio of nanocellulose whiskers/hydroxyapatite nanocomposite particles to silk fibroin is 65:35;

(5) Pour the nanocellulose whisker/hydroxyapatite/silk fibroin blend solution with the highest mass concentration prepared in step (4) into a self-made mold, and after quenching in liquid nitrogen, store it at -54 ℃ freeze-drying, and then freeze-drying other blended solutions layer by layer according to the order of mass concentration from high to low to prepare a porous bone biomimetic material with a gradient distribution of density and porosity. The bone biomimetic material is composed of carboxylated nanocellulose crystals Composed of beard, hydroxyapatite and silk fibroin, its mass ratio is 1:2:2, its density gradient from outside to inside is 78.4%, 57.4%, 11.6%, and its porosity gradient from outside to inside is respectively 17.2%, 37.4%, 80.2%.

Table 1 shows the mechanical properties and porosity of porous nano-bone biomimetic materials containing nanocellulose whiskers/hydroxyapatite/silk fibroin, the carboxymethylated nanowhiskers, nanocellulose whiskers/hydroxyphosphorus The SEM photos of the limestone composite particle structure are shown in Figure 1 and Figure 2 (the magnification is 50,000 times in Figure 1, and the magnification is 20,000 times in Figure 2). As can be seen from Figure 1, the carboxylate prepared by the present invention The average aspect ratio of the methylated nanowhiskers is about 10:1, and it can be seen from Figure 2 that only a small part of the nanowhiskers are covered by hydroxyapatite.

Example 2

A preparation method of a bone biomimetic material constructed by mineralized nano-cellulose whiskers is carried out according to the following steps:

(1) Ultrasonic disperse the nanocellulose whiskers into a solution of 1mol/L sodium hydroxide in ethanol (85%), the bath ratio is 1:500, alkalinize at 50°C for 20min, and then add 1.25mol/L chlorine Sodium acetate solution, ultrasonically stirred at 50°C for 100min, and adjusted the pH value of the solution to neutral with 0.3mol/L hydrochloric acid, and dialyzed in distilled water for three days to obtain a suspension solution of carboxylated modified nanocellulose whiskers;

(2) According to the ratio of the amount of calcium and phosphorus substances being 1.67, 0.1mol/L Ca(OH) ₂ and diluted chitosan and H ₃ PO ₄ solutions were dropped into step (1) simultaneously with a burette to obtain solution and stirred in ultrasonic for 2h, adding ammonia water dropwise to control the reaction pH to 8.0-10.0, and control the reaction temperature to 75°C to obtain a composite particle suspension with a nanocellulose whisker/hydroxyapatite ratio of 1:2. turbid liquid;

(3) Leave the suspension of nanocellulose whiskers/hydroxyapatite nanocomposite particles obtained in step (2) at room temperature for 48 hours, remove the supernatant, centrifuge to separate the precipitate, wash with distilled water three times, and freeze-dry Prepare nano-cellulose whiskers/hydroxyapatite composite particles;

(4) Ultrasonic disperse nanocellulose whiskers/hydroxyapatite composite particles obtained in step (3) into silk fibroin solutions with concentrations of 10%, 40%, and 60%, respectively, to obtain nanocellulose crystals with different concentrations. Whiskers/hydroxyapatite/silk fibroin blend solution, wherein the mass ratio of nanocellulose whiskers/hydroxyapatite nanocomposite particles to silk fibroin is 65:35;

(5) Pour the nanocellulose whisker/hydroxyapatite/silk fibroin blend solution with the highest mass concentration prepared in step (4) into a self-made mold, and after quenching in liquid nitrogen, place it at -60 ℃ freeze-drying, and then freeze-drying other blended solutions layer by layer according to the order of mass concentration from high to low to prepare a porous bone biomimetic material with a gradient distribution of density and porosity. The bone biomimetic material is composed of carboxylated nanocellulose crystals Composed of beard, hydroxyapatite and silk fibroin, the mass ratio is 1:4:3, the density gradients from the outside to the inside are 79%, 61%, and 120%, respectively, and the porosity gradients from the outside to the inside are respectively 18.1%, 26.4%, 77.2%, the mechanical properties and porosity of the prepared bone biomimetic materials are shown in Table 1, and the SEM photos of the structure of nanocellulose whiskers/hydroxyapatite composite particles are shown in Figure 3 (in the figure The magnification is 20,000 times), and it can be seen from Figure 3 that the carboxymethylated nanowhiskers are almost covered by hydroxyapatite.

Example 3

A preparation method of a bone biomimetic material constructed by mineralized nano-cellulose whiskers is carried out according to the following steps:

(1) Ultrasonically disperse nanocellulose whiskers into 0.08mol/L sodium hydroxide ethanol (85%) solution with a bath ratio of 1:500, alkalinize at 40°C for 30min, and then add 0.1mol/L Sodium chloroacetate solution was ultrasonically stirred at 55°C for 60 minutes, and the pH value of the solution was adjusted to neutral with 0.2mol/L hydrochloric acid, and dialyzed in distilled water for three days to obtain a carboxylated modified nanocellulose whisker suspension solution;

(2) According to the ratio of calcium and phosphorus substances being 1.67, drop 0.03mol/L Ca(OH) ₂ and diluted H ₃ PO ₄ solution into the solution prepared in step (1) simultaneously with a burette and ultrasonically Stir in medium for 3 hours, add ammonia water dropwise to control the reaction pH to 8.0-10.0, and control the reaction temperature to 80°C to obtain a composite particle suspension with a nanocellulose whisker/hydroxyapatite ratio of 1:4;

(3) Leave the graphene/hydroxyapatite nanocomposite particle suspension obtained in step (2) at room temperature for 48 hours, remove the supernatant, centrifuge to separate the precipitate, wash with distilled water three times, and freeze-dry to obtain nano Cellulose whiskers/hydroxyapatite composite particles;

(4) Ultrasonic disperse nanocellulose whiskers/hydroxyapatite composite particles obtained in step (3) into silk fibroin solutions with concentrations of 15%, 45%, and 65%, respectively, to obtain nanocellulose crystals with different concentrations. Whiskers/hydroxyapatite/silk fibroin blend solution, wherein the mass ratio of nanocellulose whiskers/hydroxyapatite nanocomposite particles to silk fibroin is 65:35;

(5) Pour the nanocellulose whisker/hydroxyapatite/silk fibroin blend solution with the highest mass concentration prepared in step (4) into a self-made mold, and after quenching in liquid nitrogen, store it at -75 ℃ freeze-drying, and then freeze-drying other blended solutions layer by layer according to the order of mass concentration from high to low to prepare a porous bone biomimetic material with a gradient distribution of density and porosity. The bone biomimetic material is composed of carboxylated nanocellulose crystals The mass ratio of whiskers, hydroxyapatite and silk fibroin is 1:8:6, and the density gradients from outside to inside are 81.6%, 63.2%, and 24.9%, respectively, and the porosity gradients from outside to inside are respectively 19.4%, 37.5%, and 75.3%, the mechanical properties and porosity of the prepared bone biomimetic materials are shown in Table 1, and the SEM photos of the structure of nanocellulose whiskers/hydroxyapatite composite particles are shown in Figure 4 (Fig. The magnification is 20,000 times), as can be seen from Figure 4, nanocellulose whiskers have not been observed in the field of view, and the scale of hydroxyapatite has a length of 50-300 nanometers and a width of 10-50 nanometers.

Table 1 Properties of bone biomimetic materials constructed from mineralized nanocellulose whiskers

Therefore, the present invention obtains a bone biomimetic material constructed from mineralized nanocellulose whiskers, which has a large number of interconnected nano-to-micro-scale micropores in structure, and has a gradient porosity, fully biomimetic in natural bone. Distribution structure of compact bone and cancellous bone. In terms of composition, silk fibroin with good biocompatibility and functionally optimized carboxymethylated nanocellulose whiskers are selected to enhance the compressive strength and toughness of hydroxyapatite. The bone bionic material prepared by the method of the invention has good mechanical properties, good formability and reasonable porosity, and can be used as an implantable degradable bone repair material.

Claims (7)

1. the bone biomimetic material built by the nano-cellulose whisker of mineralising, it is characterized in that: it is made up of inorganic carboxylated nano-cellulose whisker and hydroxyapatite and organic fibroin, described inorganic constituents carboxy methylation nano whisker and the mass ratio of hydroxyapatite are 1:0-1, and its mass ratio of described inorganic constituents and organic principle is 1:0.5-2.

2. the bone biomimetic material built by the nano-cellulose whisker of mineralising according to claim 1, it is characterized in that: described bone biomimetic material is made up of carboxylated nano-cellulose whisker, hydroxyapatite and Bombyx mori Silk Fibroin, the mass ratio of carboxylated nano-cellulose whisker, hydroxyapatite and Bombyx mori Silk Fibroin is 1:1-10:0.5-5, its density gradient is respectively 75.3%-82.3% from outside to inside, 57.4%-67.2%, 11.6%-30.6%.

3. the bone biomimetic material built by the nano-cellulose whisker of mineralising according to claim 1, it is characterized in that: the nanometer micropore be interconnected containing Gradient distribution in described bone biomimetic material, the porosity gradient of nanometer micropore is respectively 17.7%-24.7% from outside to inside, 32.8%-42.6%, 69.4-88.4%.

4. the bone biomimetic material built by the nano-cellulose whisker of mineralising according to claim 1, it is characterized in that: the draw ratio of described carboxylated nano-cellulose whisker is about 20:1-10:1, described fibroin albumen is Bombyx mori Silk Fibroin or tussah silk peptide, the intrinsic viscosity of its protein molecular is more than or equal to 0.50, described hydroxyapatite presents needle-like distribution, its particle length is at 50-300nm, and particle width is at 10-50nm.

5. the bone biomimetic material built by the nano-cellulose whisker of mineralising according to claim 1, is characterized in that: described hydroxyapatite and the self assembly of nano-cellulose whisker form compound particle.

6. the preparation method of the bone biomimetic material built by the nano-cellulose whisker of mineralising as described in claim 1 or 2 or 3 or 4 or 5, is characterized in that carrying out according to the following steps:

(1) by nano-cellulose whisker ultrasonic disperse in the alcoholic solution of 0.1-2mol/L sodium hydroxide; bath raio is 1:500; alkalize 10-120min at 20-80 DEG C; add the sodium chloroacetate solution of 0.125-2.5mol/L again; ultrasonic agitation 15-100min at 30-75 DEG C; and adjust solution ph to neutral and dialyse three days in distilled water with the hydrochloric acid of 0.1-0.5mol/L, obtains the brilliant suspension solution of nano-cellulose of carboxylated modification;

(2) be 1.67 according to the ratio of the amount of calcium phosphorus substance, by the Ca (OH) of 0.01-0.1mol/L ₂with dilution H ₃pO ₄solution burette to instill in the obtained solution of step (1) and at ultrasonic middle stirring 1-6h simultaneously, dropwise adding ammonia control pH value in reaction is 8.0 ~ 10.0, control reaction temperature is 20-80 DEG C, obtains the compound particle suspension that nano-cellulose whisker/hydroxyapatite ratio is 1:1-10;

(3) supernatant is removed after the nano-cellulose whisker of step (2) gained/hydroxyapatite nano-complex particle suspension room temperature being left standstill 48 hours, centrifugalize goes out precipitate, after distilled water wash three times, lyophilization obtains nano-cellulose whisker/hydroxyapatite compound particle;

(4) the nano-cellulose whisker that obtains/hydroxyapatite compound particle ultrasonic disperse in step (3) is respectively 5%-15% to concentration, 35%-45%, the organic silk cellulose solution of 55%-65%, obtain the nano-cellulose whisker/hydroxyapatite/fibroin blend solution of variable concentrations, wherein the mass ratio of nano-cellulose whisker/hydroxyapatite nano-complex particle and fibroin is 65:35;

(5) nano-cellulose whisker/hydroxyapatite/fibroin blend solution the highest for mass concentration obtained in step (4) is poured in homemade mould, after the quenching of liquid nitrogen condition, in-50--80 DEG C of lyophilization, again by other blend solution according to mass concentration order successively lyophilization from high to low, the porous bone biomimetic material of obtained density and porosity distribution gradient.

7. the preparation method of the bone biomimetic material built by the nano-cellulose whisker of mineralising according to claim 6, is characterized in that: the volumetric concentration of described alcoholic solution is 85%.