CN1166414C

CN1166414C - Method for preparing degradable biologically active artificial bone

Info

Publication number: CN1166414C
Application number: CNB021145210A
Authority: CN
Inventors: 孙明林; 李涤尘
Original assignee: Xian Jiaotong University
Current assignee: SUZHOU BC TECHNOLOGIES CO., LTD.
Priority date: 2002-04-17
Filing date: 2002-04-17
Publication date: 2004-09-15
Anticipated expiration: 2022-04-17
Also published as: CN1377711A

Abstract

The present invention discloses a method for preparing degradable biologically active artificial bones. A liquid phase deposition reaction is used under an acid condition to prepare a superfine high-purity CPC solid phase component, and a high temperature solid-solid phase reaction is used to generate a CPC solid phase system. BMP is compounded into CPC by a liquid phase compounded method to protect the bioactivity of the BMP and to make the BMP homogeneously distributed into the solidified CPC and slowly released with the formation of new bones and the degradation of materials after being transplanted in vivo. The observation of a scanning electron microscope discovers that holes between the crystals of the materials are enlarged after the BMP is compounded, which is favorable for the growth of the new bones and performs the function of promoting the degradation of the materials. Because the CPC is converted into biologically active materials with the strong function of inducing bone formation from inorganic materials by the BMP, the local metabolism of an organism is activated and characterized in the formation of the new bones. Experimental observation proves that the speed of degradation is obviously accelerated and shows that the present invention has excellent clinical application prospect. The present invention can be used for treating clinical fracture, bone disconnection, bone defect, osteoporosis, etc. and can also be used in the veterinary medicine accompanied with the treatment of bone fracture.

Description

A kind of preparation method of degradable biologically active artificial bone

One, technical field

The present invention relates to a kind of bone renovating material that clinical medicine is used, particularly a kind ofly be used for diseases such as clinical fracture, bone does not connect, bone be damaged, and to can be used for veterinary be the preparation method of the degradable biologically active artificial bone of carrier with the calcium phosphate bone cement.

Two, background technology

Ideal bone grafting material should possess following characteristics: (1) has excellent biological compatibility.(2) certain stereochemical structure and mechanical strength are arranged.(3) can be absorbed fully, degradation speed is suitable.(4) easy to use, can mouldingly arbitrarily maybe can inject use.(5) wide material sources, cheap.At present existing multiple material is used as the artificial bone graft, and as metal material, bioceramic, hydroxyapatite etc., but all there is different shortcomings in these material great majority, are difficult to satisfy fully requirements for clinical application.As the intensity and the good biocompatibility of metal and ceramic-like materials, but can not degrade; The macromolecule polymer can produce chronic reaction with body, occurs aseptic inflammation after the degraded; Other biological material such as collagen etc. lack essential space structure and mechanical strength.(Calcium phosphate cement CPC) is novel hydroxyapatite (HA) class artificial bone to calcium phosphate bone cement, compares with traditional HA, and the crystal transforming degree after its crystallization is lower, thereby to be considered to be the hydroxyapatite of non-ceramic mould.But CPC does not have the induced osteogenesis effect, but by the bone conduction effect skeletonization, thus osteogenic ability a little less than.In addition, the degradation property of CPC is not ideal enough, and is obviously slower than biological material such as allograph bone, is unfavorable for repairing the new bone remodeling in later stage when being used for the treatment of large segmental bone defect and reinvents shape.

Three, summary of the invention

According to defective or the deficiency that above-mentioned prior art partly exists, the object of the invention is, a kind of preparation method of degradable biologically active artificial bone is provided.

To achieve these goals, the present invention is according to the tissue engineering principle, synthetic voluntarily a kind of calcium-phosphorus ratio and the very approaching calcium-phosphate cement (CPC) of biological bone, and with it (Bonemorphogenetic protein, slow-released carrier BMP) are developed into the CPC/BMP cmposite artificial bone with biologic activity as bone morphogenetic protein.Design of the present invention is, adopts the acid condition highly purified CPC solid phase of liquid-phase precipitation prepared in reaction super-refinement composition down, and adopt high temperature solid-solid state reaction generation CPC solid system, BMP evenly is compounded in the inside of CPC by the liquid phase composite methods.

The technical scheme that the present invention takes is: the preparation method of degradable biologically active artificial bone, carry out by the following method:

1) utilizes the method for prior art, adopt dalcium biphosphate, tricalcium phosphate, tetracalcium phosphate and fluor-apatite in the liquid-phase precipitation prepared in reaction CPC solid phase composition under the acid condition.

2) according to the calculating of calcium phosphorus ratio, accurately take by weighing mentioned component, being mixed with calcium/phosphorus atoms ratio is 1.75: 1 mixture, carries out high temperature after the mixing and consolidates-solid state reaction, condition is 900 ℃～1200 ℃ sintering temperature 2h～4h, makes the CPC solid phase composition of super-refinement after the reaction.

3) be solvent with the distilled water, be made into the consolidation liquid of the phosphoric acid solution of 0.05mmol/L as CPC.

4) get the pure product of BMP of gene engineering expression, with the consolidation liquid dissolving, concentration is 4mg/ml～10mg/ml.If the BMP that extracts from animal bone owing to can not dissolve, then soaks with consolidation liquid, make suspension through grinding, concentration is 10mg/ml～50mg/ml.

5) take by weighing the CPC powder, with the consolidation liquid mixing that contains BMP CPC is cured, liquid phase: the solid phase ratio is (0.4～0.45) mL: 1g.

6) curing reaction carries out making finished product behind the 30min.

Research and experimental result show that this composite has biology and mechanical property preferably, and the bone repair ability is apparently higher than simple CPC.And because the adding of BMP has improved the microstructure of CPC material internal, also having activated simultaneously with new bone formation is the local metabolism of body of feature, thereby degradation speed obviously accelerates, and demonstrates the good clinical application prospect.

The present invention utilizes consolidation liquid dissolving BMP, can protect the biologic activity of BMP, and make BMP can the CPC after curing in uniform distribution.Make inorganic material possess biological action, can obviously improve the bone repair ability of material with bone-inducting active.Simultaneously, scanning electron microscopic observation finds that hole increasing between the crystal of material not only is beneficial to new bone and grows into behind the compound BMP, also plays facilitation for the degraded of material.Along with the formation of new bone and the progressively degraded of material, the BMP that is compounded in CPC inside is released slowly, therefore, the release of BMP almost is accompanied by the overall process of new bone formation and bone defect repair, and result of the test shows that this CPC/BMP cmposite artificial bone has better bone repair ability than simple CPC.Can be used for that clinical fracture, bone does not connect, bone are damaged, the treatment of osteoporosis diseases, and can be used in the veterinary with treatment of fractures.

Four, description of drawings

Fig. 1 is the photo of the CPC of the present invention after observed curing under the brilliant scanning electron microscope;

Fig. 2 is the CPC photo of the present invention behind the observed BMP of being compounded with under the brilliant scanning electron microscope;

Fig. 3, Fig. 4, Fig. 5 examine under a microscope the CPC/BMP material to implant the bone-inducting active of mice and the picture of degradation characteristic;

Fig. 6 be X line film making observe the CPC/BMP cmposite artificial bone implant the bone of rabbit damaged after, the picture of new bone formation and material degradation characteristic.

Five, the specific embodiment

The present invention is described in further detail below in conjunction with embodiment.

The inventor has provided following embodiment, but is not limited to these embodiment.

Embodiment 1: according to technical scheme of the present invention, utilize the method for prior art, adopt dalcium biphosphate, tricalcium phosphate, tetracalcium phosphate and fluor-apatite in the liquid-phase precipitation prepared in reaction CPC solid phase composition under the acid condition.Be prepared the CPC/BMP cmposite artificial bone again, carry out by the following method:

1) get different solid phase powders, being mixed with calcium/phosphorus atoms ratio according to calculating is 1.75: 1 mixture.It is standby to take by weighing this mixture 3g.

2) take by weighing the cattle BMP50mg of artificial extraction, add the phosphoric acid solution 1.5ml of 0.05mmol/L, placed 8 hours for 4 ℃, make the BMP dry product softening.Grind to form suspension under the aseptic condition.

3) with solid-phase mixture and the consolidation liquid mixing that contains BMP, reconcile into pasty state, room temperature is placed 30min and is made the CPC/BMP cmposite artificial bone.

4) similar approach is made simple CPC with the consolidation liquid that does not contain BMP.

5) with CPC and the surperficial metal spraying of CPC/BMP difference, scanning electron microscope is observed the micromorphologic variation of compound BMP front and back material surface down.As seen the CPC after solidifying is made of irregular flat, graininess and rhabdolith, and crystal is interconnection to be adhered to, with biological bone exhaustion be close.Be full of even and irregular hole between crystal, the about 20 μ m of pore size～100 μ m (referring to Fig. 1).The CPC that is compounded with behind the BMP is made of the crystal of above-mentioned form equally, adhere to a large amount of spheroidal BMP granules on the crystal and between hole equably, size is 5 μ m～10 μ m, and the simple CPC in aperture obviously increases between crystal, be about 100 μ m～500 μ m (referring to Fig. 2), be beneficial to very much new bone and grow into.The adding of this explanation biotic factor has obviously improved the microstructure of CPC material internal.

Embodiment 2: prepare CPC and CPC/BMP cmposite artificial bone according to method of the present invention, drainage is measured two kinds of porositys, and after the result showed compound BMP, the porosity of material rose to 44.5% by original 39.0%.Energy dispersive x ray analysis (energy dispersion analysis X-ray, EDAX) method is measured the calcium-phosphorus ratio of two kinds of materials, and the result shows and is 1.75: 1.The compound not influence of calcium phosphorus ratio to material of BMP is described.

Embodiment 3: prepare the CPC/BMP cmposite artificial bone according to method of the present invention, implant in the mice thigh portion flesh bag, the postoperative different time is drawn materials, fixing back section statining, and microscopically is observed the bone-inducting active and the degradation characteristic of material.Result: CPC/BMP has a large amount of chondrocytes to form after implanting for 1 week, is bulk, and the more outgrowth mesenchymal cell (referring to Fig. 3) that breaking up is arranged on every side; Chondrocyte breaks up to woven bone after 2 weeks, and the woven bone and the chondrocyte group that can see many naiveties divide a word with a hyphen at the end of a line mutually, have a spot of fibrous tissue to cut apart (referring to Fig. 4) between woven bone and the material; Woven bone changes to trabecular bone after 4 weeks, has the hemopoietic myeloid tissue to occur, and still has a small amount of new life's chondrocyte group; Cut apart by thicker fibrous tissue between material and the area of new bone after 8 weeks, it is inhomogeneous that material becomes, and disintegration phenomenon is arranged, the inner crumby granule (referring to Fig. 5) that occurs; The new bone in 16 week backs is ripe, and material is divided into little agglomerate.

Embodiment 4: prepare the CPC/BMP cmposite artificial bone according to method of the present invention, implant in the damaged model of rabbit radius 15mm bone, observe postoperative different time bone defect repair situation.The result: postoperative 2 all materials are connected closely with bone stump, and boundary is clear, and X-ray film does not have obviously new osteoplastic performance, and material volume and density do not have significant change; 4 week back materials are fuzzy with bone intersection boundary, low-density shadow occurs, are being partly dissolved due to the absorption of the cartilage of new formation and material; The material volume of 8 weeks back implantation diminishes, and marginal density reduces, and it is obvious to absorb signs of degradation; The a large amount of formation and calcifications of the new bones in 16 week backs can be seen seriality periosteum callus, and new bone is grown into to material internal by two ends, and material organically combines, the damaged scope of bone diminish (referring to Fig. 6).

Concrete application example 1: the middle humerus comminuted fracture is damaged with bone, as follows with this repair materials therapeutic process: manual traction resets, use suitable internal fixation or external plaster fixation, according to the damage range size; get this material 5ml～50ml implantable bone defect; postoperative is after 4～6 weeks, and union of fracture is good, bone is damaged to obtain the first phase reparation.Show this material accelerating bone healing speed effectively, improve new bone mass, the generation of prevention bone delay in healing or bone does not connect.

Concrete application example 2: long bone of limbs open fracture postoperative generation bone does not connect, surgical incision, the two ends fibrous tissue of will fracturing is struck off, pulp cavity is opened wide, with implanting this material after the reduction of the fracture and between the fracture end, use suitable mode to fix, conventional method is closed wound.Clinic trial confirms that this material not only can promote union of fracture in this type of trauma care, treats bone does not connect effectively and takes place.

Claims

1. the preparation method of a degradable biologically active artificial bone, adopt the highly purified calcium-phosphate cement solid phase of the liquid-phase precipitation prepared in reaction super-refinement composition under the acid condition, it is characterized in that, and adopt high temperature to consolidate-solid state reaction generation calcium-phosphate cement solid system, bone morphogenetic protein evenly is compounded in the inside of calcium-phosphate cement by the liquid phase composite methods;

Concrete preparation is carried out according to the following steps:

1) utilizes dalcium biphosphate, tricalcium phosphate, tetracalcium phosphate and fluor-apatite in the liquid-phase precipitation prepared in reaction calcium-phosphate cement solid phase composition under the acid condition of prior art;

2) according to the calculating of calcium phosphorus ratio, accurately take by weighing mentioned component, being mixed with calcium/phosphorus atoms ratio is 1.75: 1 mixture, carries out high temperature after the mixing and consolidates-solid state reaction, condition is 900 ℃～1200 ℃ sintering temperature 2h～4h, makes the calcium-phosphate cement solid phase composition of super-refinement after the reaction;

3) be solvent with the distilled water, be made into the consolidation liquid of the phosphoric acid solution of 0.05mmol/L as calcium-phosphate cement;

4) get the pure product of bone morphogenetic protein of gene engineering expression, with the consolidation liquid dissolving, concentration is 4mg/ml～10mg/ml; If the bone morphogenetic protein that extracts from animal bone owing to can not dissolve, then soaks with consolidation liquid, make suspension through grinding, concentration is 10mg/ml～50mg/ml;

5) take by weighing the calcium phosphate bone cement powder, with the consolidation liquid mixing that contains bone morphogenetic protein calcium-phosphate cement is cured, liquid phase: the solid phase ratio is 0.4～0.45mL: 1g;

(6) curing reaction carries out making finished product behind the 30min.