CN102008752A - Porous biphasic calcium phosphate biological scaffold with nano hydroxyapatite coating and preparation method thereof - Google Patents

Abstract

The invention discloses a porous biphasic calcium phosphate biological support with a nano-hydroxyapatite coating and a preparation method thereof. First, prepare ammonium dihydrogen phosphate and calcium nitrate tetrahydrate solutions respectively; mix ammonium dihydrogen phosphate solution and binder polyvinyl alcohol, heat and neutralize, put into porous biphasic calcium phosphate bioscaffold; calcium nitrate tetrahydrate solution Add it to the above reaction system, oscillate and mix well; then transfer the obtained reaction system into a reaction kettle, heat and react at 120°C for 12 hours; filter with suction, wash repeatedly with absolute ethanol and double distilled water, and dry at room temperature. The invention improves the biocompatibility of the biological support material, is beneficial to the rapid growth of cells, and also improves its clinical use value.

Description

A porous biphasic calcium phosphate bioscaffold with nano-hydroxyapatite coating and its preparation

technical field

The invention relates to the technical field of biomedical engineering, in particular to a method for preparing a nano-hydroxyapatite coating on a porous biphasic calcium phosphate biological support based on a chemical precipitation method and the obtained biological support.

Background technique

The treatment of bone defects is one of the difficult problems faced by orthopedic clinicians. Autologous bone grafting is the recognized gold standard, but the size and shape of the grafted bone are limited, and it often brings complications in the donor site, which limits its application. Transplantation is also desirable, but may spread disease and cause an immune response. Although bone cement is easy to inject and does not spread diseases, it often leads to a decrease in local bone strength, fatal allergic reactions, and heat production because it does not have the ability to regenerate bone. These methods all have their own shortcomings, and it is difficult to meet the needs of clinical bone defect repair. The emergence of tissue-engineered bone provides a new idea to overcome the above-mentioned shortcomings. Its basic idea is to combine living cells with scaffold materials to form bone substitutes after cultured and proliferated in vitro. An ideal scaffold material for bone tissue engineering should have the following characteristics: 1. Good osteoconductivity, with a three-dimensional porous structure; 2. Good biocompatibility; 3. Good biodegradability, the material itself and its degradation products are Harmless to the human body; 4. Has a certain mechanical strength; 5. Easy to shape. Currently used scaffold materials mainly include: natural polymer materials mainly include collagen (Col), acellular matrix (ACTM), chitin (CS) and its derivative chitosan (also known as chitosan), fibrin, etc. Good biocompatibility, conducive to cell adhesion, proliferation and differentiation. Synthetic polymer materials mainly include polyglycolic acid (PGA), polylactic acid (PLA), polycaprolactone (PCL), polyethylene lactide copolymer (PLGA), polymethyl methacrylate (PMMA), etc. Hydroxyapatite (HA) is the main inorganic component of biological hard tissues. It has a lamellar structure and nanocrystalline characteristics. It not only has good biocompatibility and non-toxicity, but also conducts bone growth. It forms a chemical bond with the tissue on the interface. Once the cells attach and stretch, they can produce bone matrix collagen, and then further mineralize to form bone tissue; but there are some shortcomings in simply using HA as a load-bearing component of the human body, such as low strength and elasticity. High modulus, high brittleness, poor toughness and unsatisfactory molding. β-TCP has good biodegradability, compatibility and non-toxicity. When it is implanted in the human body, it can guide the growth of new bone. The degraded calcium and phosphorus can enter the living body's circulatory system to form new bone, which is better for bone induction. However, its shortcoming is that its degradation rate is too fast, which is not conducive to bone healing and cell attachment. Compared with single-phase bioceramics (HA or β-TCP), biphasic bioceramics, which are mixed with different proportions of HA and β-TCP, have more suitable biodegradability and are beneficial to induce osteogenesis. Ramay et al. developed a nano-biphasic porous calcium phosphate scaffold, which is composed of TCP matrix and nano-hydroxyapatite. Its compressive strength is similar to that of cancellous bone and its porosity is 73%. In recent years, with the continuous development of nanometer knowledge and technology, it has been found that hydroxyapatite in human bones is mainly a nanoscale needle-like single crystal structure. Nanoscale hydroxyapatite is more similar to the composition of bone tissue in the human body and has better biocompatibility. According to the "nano effect" theory, the surface area of nanoparticles per unit mass is significantly larger than that of micron-sized particles, which significantly increases the number of atoms on the surface of the particles and improves the activity of the particles, thereby facilitating tissue bonding and mechanical properties (strength, toughness) and superplasticity). Compared with ordinary HA, nano-HA showed a higher cell proliferation rate, and with the decrease of the concentration of the extract and the increase of the culture time, the cytotoxicity tended to be zero; the direct contact between the micro-particles of the nano-HA and the cells did not See cytotoxicity, easy to attach and grow with cells, and better biocompatibility. The induced osteogenesis of artificial bone is affected by the void structure of the material. Studies have found that a larger pore size of 400-800 microns is more conducive to the formation of new bone than a small pore size, while calcium phosphate ceramics with a pore size of less than 150 microns and a porosity of less than 30% The ingrowth of new bone is significantly restricted.

Nano-hydroxyapatite has better biocompatibility and is conducive to cell adhesion. Using nano-hydroxyapatite coating to modify the surface of biological materials to make them biologically active is one of the research directions. The current preparation methods of nano-hydroxyapatite coating include plasma spraying method, laser cladding method, electrophoretic deposition method, electrochemical deposition method, bionic method and so on. And most of them focus on the research on the preparation of nano-nano hydroxyapatite coating on the metal surface. The research on modifying the surface of inorganic substances to make them have better biocompatibility and bioactivity has been reported at home and abroad. The present invention adopts the chemical method to produce nano-hydroxyapatite, directly in the pores of porous biphasic calcium phosphate biological support (HA/β-TCP=6-7/3-4, porosity 40-60%, aperture 100-500 microns) Nano-hydroxyapatite coatings are prepared inside and on the surface to improve the biocompatibility of biological scaffold materials, facilitate the rapid growth of cells, and improve their clinical use value.

Contents of the invention

The purpose of the present invention is to provide a method of "coating" nano-hydroxyapatite in the pores and surfaces of porous biphasic calcium phosphate biostents, and such a bioscaffold obtained to improve the biocompatibility of bioscaffold materials It is beneficial to the rapid growth of cells and improves its clinical value.

A porous biphasic calcium phosphate biological support with nanometer hydroxyapatite coating, the outer surface and void surface of the biological support are uniformly distributed with 100% (100% refers to purity) hydroxyapatite short rod crystals.

The short rod-like crystals of hydroxyapatite have a diameter of 20-30 nanometers and a length of 100-200 nanometers.

The porous biphasic calcium phosphate biological scaffold (mixture of hydroxyapatite and β-tricalcium phosphate, HA/β-TCP) HA/β-TCP mass ratio=6-7/3-4, porosity 40-60 %, pore size 100-500 microns.

A method for preparing a porous biphasic calcium phosphate bioscaffold with a nano-hydroxyapatite coating, comprising the following steps:

1) prepare ammonium dihydrogen phosphate and calcium nitrate tetrahydrate solution respectively;

2) mixing the ammonium dihydrogen phosphate solution and the binder polyvinyl alcohol, heating and neutralizing, and placing the porous biphasic calcium phosphate bioscaffold;

3) adding the calcium nitrate tetrahydrate solution into the reaction system obtained in step 2), shaking and mixing;

4) Transfer the reaction system obtained in step 3) into a reaction kettle, heat and react at 120° C. for 12 hours;

5) Suction filtration, repeated washing with absolute ethanol and double distilled water, and drying at room temperature.

The concentrations of the ammonium dihydrogen phosphate and calcium nitrate tetrahydrate solution described in step 1) are respectively 0.3mol/L and 0.5mol/L.

The molar ratio of calcium nitrate tetrahydrate: ammonium dihydrogen phosphate: PVA is 15:25:1.

The heating and neutralization in step 2) is to heat to 80-100°C, stir and mix well, adjust the pH value to 10.5-11.5 with concentrated ammonia water, and then put the porous biphasic calcium phosphate biostent

When adding the calcium nitrate tetrahydrate solution described in step 3), drop the calcium nitrate tetrahydrate solution into the reaction system obtained in step 2) at 2 ml/min under ultrasonic vibration.

Step 4) The heating reaction is to transfer the reaction system into a reaction kettle and then put it into a vacuum dryer.

The mass ratio of the porous biphasic calcium phosphate biological scaffold HA/β-TCP is 6-7/3-4, the porosity is 40-60%, and the pore diameter is 100-500 microns.

The current preparation methods of nano-hydroxyapatite coating include plasma spraying method, laser cladding method, electrophoretic deposition method, electrochemical deposition method, bionic method and so on. And most of them focus on the research on the preparation of nano-nano hydroxyapatite coating on the metal surface. The invention successfully adopts the chemical precipitation method to combine the nano-hydroxyapatite to the inner and outer surfaces of the biphasic tricalcium phosphate porous biological support in the form of "coating" to make the biological support material of the nanoHA/BCP composite material. For the first time, nano-hydroxyapatite coatings were prepared on inorganic materials (biphasic calcium phosphate), which is conducive to the direct attachment and growth of seed cells and bone marrow stromal cells on the surface of nano-hydroxyapatite, making full use of nano-hydroxyapatite Good biocompatibility and adjustable biphasic calcium phosphate degradation rate, which not only give full play to the advantages of easy adhesion and growth of cells on the surface of nano-hydroxyapatite, but also solve the problem that nano-hydroxyapatite is difficult to form, and take into account The problem of inconsistency between monophasic calcium phosphate biodegradation time and new bone formation rate was solved. The new bio-scaffold was tested on an animal spinal fusion model, and the results showed that the novel bio-scaffold was more conducive to the expansion of bone cells and the formation of new bone.

Description of drawings

Fig. 1 is the schematic diagram of the BCP support prepared by the present invention;

Fig. 2 is electron microscope scanning BCP support pore inner surface figure of the present invention;

Fig. 3 is the X-ray diffractogram of BCP scaffold composition of the present invention;

Fig. 4 is electron microscope scanning BCP stent coating pore inner surface figure of the present invention;

Fig. 5 is an X-ray diffraction diagram of the BCP stent coating composition of the present invention.

Fig. 6 is a diagram showing the osteogenesis of the scaffold material of the present invention in the space of the fusion hard tissue section of the rabbit spine.

Detailed ways

The following examples are only used to further illustrate the present invention, rather than to limit the present invention.

Example 1

1. Preparation of porous biphasic calcium phosphate bioscaffold:

Prepare hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) composite powder in a certain proportion and pass through a 200-mesh sieve, add a certain proportion of forming agent (polyvinyl alcohol, PVA), add water to make a paste, and use Polyurethane foam with appropriate pore diameter is fully impregnated, dried at a certain temperature, and then sintered at high temperature for 2-3 hours to obtain a composite porous biphasic calcium phosphate scaffold (BCP) with the same shape as the foam, uniform distribution of pores, and a pore diameter of 200-500 μm. ).

2. Prepare 0.3mol/L ammonium dihydrogen phosphate and 0.5mol/L calcium nitrate tetrahydrate with deionized water;

3. Take 25ml of the prepared ammonium dihydrogen phosphate solution, heat it at 80-100℃ with magnetic stirring and 0.0005mol PVP to fully dissolve and mix, adjust the pH to 10.5-11.5 with NH3·H2O, and put the BCP stent into the supernatant.

4. Transfer 25ml of calcium nitrate tetrahydrate solution into a medical glass bottle, and control the drip rate through an infusion set. At a water temperature of 37°C and under ultrasonic vibration, drop calcium nitrate tetrahydrate solution into ammonium dihydrogen phosphate containing BCP stent at a constant speed of 2ml/min and mix. liquid. Sonication was maintained for 30 minutes to thoroughly mix the reaction.

5. After the reaction system was transferred to the reactor, put it into a vacuum dryer at 120°C for 12 hours.

6. Negative pressure suction filtration, repeated washing with absolute ethanol and double distilled water for 2 times, and drying at room temperature to obtain short rod-shaped nano-hydroxyphosphorus with a diameter of 20-30 nanometers and a length of 100-200 nanometers on the outer surface of the biological scaffold and the inside of the hole. Greystone crystals evenly coat.

Table 1 is the X-ray diffraction diagram analysis index of the BCP support composition of the present invention (the content of tricalcium phosphate and hydroxyapatite in the BCP support of the peak height measurement method is respectively 33.9% and 66.1%; the peak area rule is 36.8% and 63.2% )

Table 1

Table 2 is the X-ray diffraction pattern analysis index of the BCP stent coating composition of the present invention (using the peak height measurement method and the peak area rule, the BCP stent coating composition is hydroxyapatite, and the purity is 100%)

Table 2

Claims (10)

1. the porous biphasic calcium phosphate biological support with nano hydroxyapatite coating is characterized in that the outer surface of described biological support and surface, space are evenly distributed with nano-grade hydroxy apatite corynebacterium crystal.

2. biological support according to claim 1 is characterized in that, described hydroxyapatite corynebacterium crystal diameter is the 20-30 nanometer, and length is the 100-200 nanometer.

3. biological support according to claim 1 is characterized in that, described porous biphasic calcium phosphate biological support HA/ β-TCP mass ratio=6-7/3-4, and voidage is 40-60%, the aperture is the 100-500 micron.

4. the preparation method with porous biphasic calcium phosphate biological support of nano hydroxyapatite coating is characterized in that, may further comprise the steps:

1) prepares Ammonium biphosphate and four water-calcium nitrate solution respectively;

2) ammonium dihydrogen phosphate and adhesive polyethylene alcohol are mixed, add thermo-neutrality, put into porous biphasic calcium phosphate biological support;

3) four water-calcium nitrate solution is added step 2) in the reaction system that obtains, the concussion mixing;

4) reaction system that step 3) is obtained changes in the reactor, 120 ℃, continues 12 hours reacting by heating;

5) sucking filtration, behind dehydrated alcohol, the distilled water cyclic washing, room temperature is dried.

5. preparation method according to claim 3 is characterized in that, the concentration of described Ammonium biphosphate of step 1) and four water-calcium nitrate solution is respectively 0.3mol/L and 0.5mol/L.

6. preparation method according to claim 3 is characterized in that, described four water-calcium nitrate: Ammonium biphosphate: the PVA mol ratio is 15: 25: 1.

7. preparation method according to claim 3 is characterized in that step 2) the described thermo-neutrality that adds is after being heated to 80-100 ℃ of stirring and evenly mixing, with strong aqua ammonia pH value to be transferred to 10.5-11.5, puts into porous biphasic calcium phosphate biological support again

8. preparation method according to claim 3 is characterized in that, during the described adding four water-calcium nitrate of step 3) solution, splashes into step 2 at following four water-calcium nitrate solution of ultrasonic concussion with 2ml/min) in the reaction system that obtains.

9. preparation method according to claim 3 is characterized in that, the described reacting by heating of step 4) is to insert vacuum drier after reaction system is changed over to reactor.

10. preparation method according to claim 3 is characterized in that, described porous biphasic calcium phosphate biological support HA/ β-TCP mass ratio=6-7/3-4, voidage 40-60%, aperture 100-500 micron.

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