CN101791437B - Preparation method of polymer/inorganic particle composite bone repair porous scaffold - Google Patents

Abstract

The invention discloses a prepration method of a polymer/inorganic particle composite bone repair porous scaffold. The process comprises the following steps: adding inorganic particles into deionized water and preparing suspension of the inorganic particles; adding molten paraffin wax into the suspension, and obtaining pickering emulsion after stirring; cooling the emulsion to room temperature, filtering, and then obtaining paraffin wax microspheres coated with the inorganic particles on the surface; mixing the paraffin wax microspheres with polymer solution, reducing the temperature, leading the polymer solution to carry out phase separation, freeze-drying, further using n-hexane for washing and removing the paraffin wax microspheres, drying and finally obtaining the polymer/inorganic particle composite bone repair porous scaffold. The prepared polymer/inorganic particle composite porous scaffold has high porosity and pore connectivity, and the inorganic particles are distributed on the pore surface of the porous scaffold, thereby having better biological activity; and the scaffold can meet the requirements of bone tissue engineering scaffold materials on pores and pore diameter structure, thereby having great application prospects.

Description

A kind of method for preparing of polymer/inorganic particle composite bone repair porous scaffold

Technical field

The present invention relates to a kind of method for preparing of polymer/inorganic particle composite bone repair porous scaffold, the paraffin ball that relates to the inorganic particulate coating specifically is the method for preparing that porogen prepares the polymer/inorganic particle complex stephanoporate bracket.

Background technology

Bone injury and bone are damaged to be present common disease, and traditional treatment is to carry out carrying out bone tissue restoration from body or allogenic bone transplantation.The autologous bone transplanting repairing effect is good, but has the shortcoming of donor deficiency, second operation and donor position complication, and there is immunogenic shortcoming in allogenic bone transplantation and the danger of pathophoresis is arranged.The technology of organizational project and regenerative medicine and method can become the live body osseous tissue through the differential growth of inducing of osteoblastic growth or stem cell, and be used for the reparation and the regeneration of bone, thereby are expected to the reparation that promotes that bone is damaged.Wherein, bone repairing support plays crucial effect in osteanagenesis.

Polymeric material has good processing properties and controlled degradation property, but lack the cytoactive site, is difficult to promote the expression of the normal activity of osteoblast.And biological activity inorganic material such as hydroxyapatite etc. have good biological activity, therefore can both advantages be combined through the compound of biological activity inorganic material and polymer, give support good processing properties, degradation property and biological activity.

The polymer/inorganic particle compound rest normally is dispersed in inorganic particulate in the polymer solution, is separated through thermic then or method preparation that particle is separated out.In the support of these method preparations, inorganic particulate is distributed in the polymeric matrix, has therefore limited the interaction of cell and biological activity inorganic particulate.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, a kind of biological activity that can effectively improve polymer is provided, promote the method for preparing of the polymer/inorganic particle composite bone repair porous scaffold of osteoblast increment and differentiation.

The method for preparing of polymer/inorganic particle composite bone repair porous scaffold may further comprise the steps:

(1) inorganic particulate is joined in the deionized water, compound concentration is the inorganic particulate suspension of 10～50mg/ml, behind the ultra-sonic dispersion, is heated to 65～80 ℃;

(2) with the hard paraffin heating, obtain fused vasoliniment;

(3) fused vasoliniment is joined in the inorganic particulate suspension; Vasoliniment is 20～30% with inorganic particulate suspension vol ratio; Stirring obtains pik woods (pickering) emulsion; Be cooled to room temperature, filter the back, obtain the paraffin microsphere of surface-coated inorganic particulate with deionized water wash, lyophilizing;

(4) with polymer dissolution in dioxane or dimethyl sulfoxide, the polymer solution of compound concentration 0.05～0.5g/ml is under the room temperature; With paraffin microsphere and the polymer solution that step (3) obtains,, insert in the mould according to the ratio uniform mixing of 0.5～3g paraffin ball/ml polymer solution; Reduce temperature then to-80～-20 ℃, polymer solution is separated, lyophilization; The demoulding obtains the paraffin microsphere and the mixture of polymers of surface-coated inorganic particulate;

(5) the paraffin microsphere and the mixture of polymers that step (4) are obtained place normal hexane to wash, and except that deparaffnize, vacuum drying, obtain the porous polymer scaffold of hole surface coating inorganic particle.

Said polymer can be polylactic acid, polyglycolic acid, polycaprolactone, poly butyric ester, polyurethane and above-mentioned mixture of polymers or the copolymer between them among the present invention.

Said inorganic particulate is a kind of in the adulterated above-mentioned inorganic particulate of hydroxyapatite, calcium phosphate, calcium silicates, bioactivity glass particle and strontium element.

Beneficial effect of the present invention is:

The paraffin microsphere that the present invention applies with inorganic particulate is as porogen; The polymer/inorganic particle complex stephanoporate bracket of preparation has high porosity and the hole is connective; Its inner hole wall is that inorganic particulate covers; Overcome in the polymer/inorganic particle complex stephanoporate bracket traditional preparation process method polymer solution and inorganic particulate have been pre-mixed the inorganic particulate that causes by the shortcoming of polymer overmold; Thereby increased the chance that osteoblast contacts with inorganic particulate, can effectively improve the biological activity of polymer support, promoted osteoblastic increment and differentiation.Therefore, this complex stephanoporate bracket has the damaged using value of very strong repair tissue.Method for preparing of the present invention is simple, material source is extensive, production efficiency is high.

Description of drawings

Fig. 1 is the stereoscan photograph of synthetic hydroapatite particles;

Fig. 2 is the stereoscan photograph of the paraffin microsphere of hydroxyapatite coating;

Fig. 3 (a) is the stereoscan photograph in the PLGA complex stephanoporate bracket cross section of surface-coated hydroxyapatite particle;

Fig. 3 (b) is the stereoscan photograph of the PLGA complex stephanoporate bracket macropore hole wall of surface-coated hydroxyapatite particle;

Fig. 4 (a) is the support cross section stereoscan photograph that the PLGA complex stephanoporate bracket of surface-coated hydroxyapatite particle soaked in simulated body fluid 4 days;

Fig. 4 (b) is the support macropore hole wall stereoscan photograph that the PLGA complex stephanoporate bracket of surface-coated hydroxyapatite particle soaked in simulated body fluid 4 days;

Fig. 5 is distribution and the form laser confocal microscope photo of osteoblast in the PLGA complex stephanoporate bracket of surface-coated hydroxyapatite particle;

Fig. 6 is distribution and the stereoscan photograph of form of osteoblast in the PLGA complex stephanoporate bracket of surface-coated hydroxyapatite particle.

The specific embodiment

Further specify the present invention below in conjunction with instance, but these instances are not used for limiting the present invention.

Instance 1:

(1) lime nitrate and diammonium phosphate are dissolved in respectively in the water, preparation 400ml concentration is that ammonium dibasic phosphate aqueous solution and the concentration of 0.3mol/L is the calcium nitrate aqueous solution of 0.5mol/L; The aqueous solution of diammonium phosphate is dropwise joined in the calcium nitrate aqueous solution, magnetic agitation, regulating pH value is 10, and reaction is 2 hours in 60 ℃ of water-baths, and ageing is 24 hours again; Use the deionized water cyclic washing, lyophilization obtains hydroxyapatite nano particle, and is as shown in Figure 1.Can be found out by figure, hydroxyapatite nano particle is a needle-like, is about 300 nanometers.

(2) the hydroxyapatite particle is joined in the deionized water, compound concentration is the suspension of 30mg/ml, and ultra-sonic dispersion is heated to 70 ℃; With the hard paraffin heating, obtain fused vasoliniment;

(3) the fused vasoliniment of 30ml is joined in the 100ml hydroxyapatite particle suspension; Stirring obtains the pickering emulsion, is cooled to room temperature, filters the back with deionized water wash, lyophilizing; Obtain the paraffin microsphere of surface-coated hydroxyapatite particle; Adopt the domestic standard sieve that the paraffin microsphere of hydroxyapatite particle coated is carried out classification, obtain the microsphere of particle diameter 450～600 μ m, as shown in Figure 2.

(4) under the room temperature, the paraffin microsphere that step (3) is obtained mixes with the dioxane solution of the polylactic-co-glycolic acid (PLGA) of concentration 0.1g/ml, inserts in the mould; The consumption of paraffin ball is every milliliter of PLGA solution of 1.3g; Said mixture is freezing under-20 ℃, polymer solution is separated, dioxane was removed in lyophilization in 6 hours; The demoulding obtains the paraffin microsphere of surface-coated hydroxyapatite particle and the mixture of PLGA;

(5) paraffin microsphere that step (4) is obtained and the mixture of PLGA place normal hexane to wash, and remove deparaffnize, and vacuum drying obtains the PLGA complex stephanoporate bracket that hole surface applies the hydroxyapatite particle.Shown in Fig. 3 (a), porous support has the macropore of 450-600 μ m, and macropore has good connectedness.The big hole surface of compound rest covers for the hydroxyapatite particle, shown in Fig. 3 (b).

The PLGA complex stephanoporate bracket of surface-coated hydroxyapatite particle of preparation is immersed in 1.5 times the simulated body fluid, puts into 37 ℃ of thermostatic water bath.Fig. 4 (a) and the PLGA complex stephanoporate bracket that (b) is respectively surface-coated hydroxyapatite particle soak 4 days the support cross section and the stereoscan photograph of punching hole wall in 1.5 times simulated body fluid.By Fig. 4 (a) and (b) visible, the PLGA complex stephanoporate bracket of surface-coated hydroxyapatite particle has good biological activity.The PLGA complex stephanoporate bracket of the surface-coated hydroxyapatite particle for preparing is put in 24 well culture plates, adds culture fluid, in incubator, carry out In vitro culture.The form of cell culture after one week adopts laser confocal microscope and scanning electron microscope to observe; The result sees Fig. 5 and Fig. 6 respectively; Visible by figure, osteoblast form on the PLGA complex stephanoporate bracket of surface-coated hydroxyapatite particle is sprawled, and is expected to the reparation that promotes that bone is damaged.

Instance 2:

(1) lime nitrate and sodium silicate are dissolved in respectively in the water, preparation 400ml concentration is that sodium silicate aqueous solution and the concentration of 0.5mol/L is the calcium nitrate aqueous solution of 0.5mol/L; The aqueous solution of sodium silicate is dropwise joined in the calcium nitrate aqueous solution, magnetic agitation, regulating pH value is 10, and reaction is 2 hours in 60 ℃ of water-baths, and ageing is 24 hours again; Use the deionized water cyclic washing, lyophilization obtains the calcium silicates particle;

(2) the calcium silicates particle is joined in the deionized water, compound concentration is the suspension of 40mg/ml, and ultra-sonic dispersion is heated to 70 ℃; With the hard paraffin heating, obtain fused vasoliniment;

(3) the fused vasoliniment of 20ml is joined in the 100ml calcium silicates particle suspension, stir and obtain the pickering emulsion, be cooled to room temperature, filter the back, obtain the paraffin microsphere of surface-coated calcium silicates particle with deionized water wash, lyophilizing;

(4) under the room temperature, the paraffin microsphere that step (3) is obtained mixes with the dioxane solution of the polylactic-co-glycolic acid (PLGA) of concentration 0.1g/ml, inserts in the mould; The consumption of paraffin ball is every milliliter of PLGA solution of 1.3g; Said mixture is freezing under-20 ℃, polymer solution is separated, dioxane was removed in lyophilization in 6 hours; The demoulding obtains the paraffin microsphere of surface-coated calcium silicates and the mixture of PLGA;

(5) paraffin microsphere that step (4) is obtained and the mixture of PLGA place normal hexane to wash, and remove deparaffnize, and vacuum drying obtains the PLGA porous support that hole surface applies calcium silicates.

Instance 3:

(1) with the step in the instance 1 (1), but the aqueous solution of 0.5mol/L lime nitrate become the mixed aqueous solution of strontium nitrate of lime nitrate and the 0.05mol/L of 0.45mol/L, prepare strontium doping hydroxyapatite particle.

(2) strontium doping hydroxyapatite particle is joined in the deionized water, compound concentration is the suspension of 40mg/ml, and ultra-sonic dispersion is heated to 70 ℃; With the hard paraffin heating, obtain fused vasoliniment;

(3) the fused vasoliniment of 20ml is joined in the 100ml strontium doping hydroxyapatite particle suspension; Stirring obtains the pickering emulsion; Be cooled to room temperature, filter the back, obtain the paraffin microsphere of surface-coated strontium doping hydroxyapatite particle with deionized water wash, lyophilizing;

(4) under the room temperature, the paraffin microsphere that step (3) is obtained mixes with the dioxane solution of the polylactic acid (PLLA) of concentration 0.5g/ml, inserts in the mould; The consumption of paraffin ball is every milliliter of PLLA solution of 3g; Said mixture is freezing under-80 ℃, polymer solution is separated, dioxane was removed in lyophilization in 6 hours; The demoulding obtains the paraffin microsphere of surface-coated strontium doping hydroxyapatite and the mixture of PLLA;

(5) paraffin microsphere that step (4) is obtained and the mixture of PLLA place normal hexane to wash, and remove deparaffnize, and vacuum drying obtains the PLLA porous support of surface-coated strontium doping hydroxyapatite.

Instance 4:

Step is with the step ofinstance 1, but what adopt is the dioxane solution of polycaprolactone, polycaprolactone/hydroxyapatite composite porous bracket.

Instance 5:

Step is with the step ofinstance 1, but what adopt is the dimethyl sulphoxide solution of polyurethane, polyurethane/hydroxyapatite composite porous bracket.

Claims (1)

1. the method for preparing of a polymer/inorganic particle composite bone repair porous scaffold, its step is following:

(1) inorganic particulate is joined in the deionized water, compound concentration is the inorganic particulate suspension of 10～50mg/ml, behind the ultra-sonic dispersion, is heated to 65～80 ℃; Said inorganic particulate is a kind of in the adulterated above-mentioned inorganic particulate of hydroxyapatite, calcium phosphate, calcium silicates, bioactivity glass particle and strontium element;

(2) with the hard paraffin heating, obtain fused vasoliniment;

(3) fused vasoliniment is joined in the inorganic particulate suspension; Vasoliniment is 20～30% with inorganic particulate suspension vol ratio, stirs and obtains pickering emulsion, is cooled to room temperature; Filter the back with deionized water wash, lyophilizing, obtain the paraffin microsphere of surface-coated inorganic particulate;

(4) with polymer dissolution in dioxane or dimethyl sulfoxide, the polymer solution of compound concentration 0.05～0.5g/ml is under the room temperature; With paraffin microsphere and the polymer solution that step (3) obtains,, insert in the mould according to the ratio uniform mixing of 0.5～3g paraffin ball/ml polymer solution; Reduce temperature then to-80～-20 ℃, polymer solution is separated, lyophilization; The demoulding obtains the paraffin microsphere and the mixture of polymers of surface-coated inorganic particulate; Said polymer is polylactic acid, polyglycolic acid, polycaprolactone, poly butyric ester, polyurethane and above-mentioned mixture of polymers or the copolymer between them;

(5) the paraffin microsphere and the mixture of polymers that step (4) are obtained place normal hexane to wash, and except that deparaffnize, vacuum drying, obtain the porous polymer scaffold of hole surface coating inorganic particle.

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