CN112023112A

CN112023112A - Bone hemostatic material with osteogenesis inducing activity and preparation method thereof

Info

Publication number: CN112023112A
Application number: CN202011002703.XA
Authority: CN
Inventors: 李湘杰; 任芳; 丁力; 王长江; 盖增; 祝君梅; 朱晓丽; 刘红云; 聂卫
Original assignee: Tianjin Zhongjin Biological Development Co ltd
Current assignee: Tianjin Zhongjin Biological Development Co ltd
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2020-12-04

Abstract

The invention provides a preparation method of a bone hemostatic material with osteogenesis inducing activity and the bone hemostatic material prepared by the preparation method, the bone hemostatic material comprises an active bone extract accounting for 0.5-5% of the bone hemostatic material by mass, a dispersing agent accounting for 10-23% of the bone hemostatic material by mass and a powder auxiliary agent accounting for 72.9-89.5% of the bone hemostatic material by mass, and the active bone extract comprises the following components in percentage by mass: blood coagulation agent: 21.3 to 62 percent; adhesive: 19.5 to 53.7 percent; a regulator: 2.7-11%; inducing osteogenesis agent: 6.1-23.4%; bacteriostatic agent: 1.4-7.5% and also provides a preparation method of the hemostatic material. The bone hemostatic material with osteogenesis inducing activity and the preparation method thereof have good adhesion, good hemostatic effect and degradability, do not cause the problems of bone wound occupation or bone wound difficult repair due to the existence of foreign matters, and have good biological safety and biocompatibility.

Description

Bone hemostatic material with osteogenesis inducing activity and preparation method thereof

Technical Field

The invention belongs to the technical field of bone hemostatic materials, and particularly relates to a bone hemostatic material with osteogenesis inducing activity and a preparation method thereof.

Background

Clinically, bone wound bleeding is difficult to stop bleeding by vasoconstriction, and conventional hemostasis methods in the operation, such as electric coagulation, forceps, hemostatic materials and the like, have poor effects. The bone hemostatic material commonly used in clinic is bone wax, which generally contains beeswax or vaseline, and the hemostatic principle is to mechanically plug and block capillary bleeding of a bone wound surface, but the material is not degradable, and once the bone hemostatic material is used in clinic, foreign matters permanently exist in a body. The foreign body occupies the bone wound surface, causes the bone wound surface to be difficult to repair and can not be well healed, and increases the risk of postoperative infection.

Researchers at home and abroad have developed degradable bone hemostatic materials such as collagen, gelatin sponges, oxidized cellulose, polylactic acid, polysaccharide and the like from various aspects. Although these materials are degradable, the adhesion of the materials is far less than that of the traditional bone wax, and the materials are easily washed away by blood flow during operation, so that the expected hemostatic effect cannot be achieved. Moreover, most of the materials have biocompatibility which cannot meet the requirements of bone implant materials, and the biological safety problems of cytotoxicity, blood compatibility, pyrogen reaction and the like exist. Therefore, no ideal hemostatic material for orthopedics department exists at present.

Disclosure of Invention

In view of the above, the present invention provides a bone hemostatic material with osteogenesis inducing activity and a preparation method thereof, which has good adhesion, good hemostatic effect, degradability, no occurrence of bone wound occupation or bone wound difficult repair due to the presence of foreign substances, and good biosafety and biocompatibility.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a preparation method of a bone hemostatic material with osteogenesis inducing activity comprises the following steps:

(1) crushing: crushing cortical bone blocks at low temperature to obtain cortical bone particles;

further, the crushing process is preferably to crush the cortical bone pieces into cortical bone granules of 100 mesh to 6 mesh by keeping the temperature low with liquid nitrogen.

(2) Degreasing: placing the cortical bone grains in degreasing liquid for degreasing to obtain degreased cortical bone grains;

further, the degreasing solution is a chloroform/methanol mixed solution with the volume ratio of 1: 1.

Further, the degreasing step is that methanol and chloroform which are 0.1-0.6 time and 0.2-2 times of the weight of the cortical bone particles are added into a reaction tank in sequence and react for 12-24 hours at room temperature.

(3) Decalcification: soaking the degreased cortical bone particles obtained in the step (2) in a decalcifying solution to obtain decalcified cortical bone particles;

further, the decalcification solution is 0.4-0.6mol/L hydrochloric acid.

Further, the decalcification step is to soak the degreased cortical bone grains in a decalcification solution, wherein the decalcification solution just submerges the cortical bone grains, and the reaction is carried out at room temperature for no less than 24 hours.

(4) Deproteinization: sequentially soaking the decalcified cortical bone particles obtained in the step (3) in a calcium chloride solution, an EDTA & 2Na solution, a lithium chloride solution and water to obtain deproteinized cortical bone particles;

further, the deproteinization step is to soak the decalcified cortical bone particles in 2-4mol/L calcium chloride solution, the calcium chloride solution submerges the cortical bone particles, and the reaction is carried out for at least 10h at room temperature; then taking out the cortical bone particles, soaking the cortical bone particles in 0.4-0.8mol/L EDTA & 2Na solution, wherein the EDTA & 2Na solution needs to cover the cortical bone particles, and reacting for at least 8h at room temperature; then taking out the cortical bone particles, soaking the cortical bone particles in 6-10mol/L lithium chloride solution, wherein the lithium chloride solution is required to submerge the cortical bone particles, and reacting for at least 8h at room temperature; finally, the cortical bone grains are taken out, soaked in water until the water submerges the cortical bone grains, and reacted for 5 hours at the temperature of 50-60 ℃.

(5) Extraction: placing the deproteinized cortical bone particles obtained in the step (4) into an extract for reaction, then concentrating the extract, and dialyzing the concentrated extract;

further, the preparation method of the extract comprises the following steps: adding 4-8mol/L urea solution, 0.4-0.8mol/L calcium chloride solution and 0.005-0.02 mol/L NEMI into purified water, fully stirring until the solution is dissolved, and diluting to 1L to obtain the extract.

And (3) further, adding the deproteinized cortical bone particles obtained in the step (4) into an extraction solution, reacting for 12-18 h at room temperature, concentrating the extraction solution by using an ultrafiltration machine, pouring the concentrated extraction solution into a container separated by a semipermeable membrane, wherein the semipermeable membrane is purified water and the extraction solution at one side, and dialyzing for not less than 50h at room temperature.

(6) Centrifugal drying: centrifuging the dialyzed extractive solution, dispersing the precipitate with dispersion liquid, dialyzing at room temperature, centrifuging the dialyzed mixture, and drying the precipitate to obtain active bone extract.

Further, the centrifugal drying step is to centrifuge the dialyzed extract, the centrifugation speed is 15000RPM to 20000RPM, the centrifugation time is 30min to 45min, the precipitate obtained by centrifugation is dispersed by water and then placed in a container separated by a semipermeable membrane, the semipermeable membrane is a bone extract dispersion liquid while the semipermeable membrane is a buffer solution of 0.2mol/L citric acid/0.05 mol/L sodium citrate, and the dialysis is carried out for 10h to 24h at room temperature. And centrifuging again after dialysis, wherein the centrifugation speed is 3000 RPM-40000 RPM, the centrifugation time is 30min-45min, and the centrifuged precipitate is dried to obtain the active bone extract.

(7) And (4) mixing the active bone extract obtained in the step (6) with a dispersing agent and a powder auxiliary agent to obtain the bone hemostatic material with the activity of inducing osteogenesis.

Further, the dispersing agent is preferably one or a mixture of more of soybean lecithin, egg yolk lecithin, vitamin E, soybean oil and soybean lecithin.

Furthermore, the powder auxiliary agent is one or a mixture of more of hydroxyapatite, calcium phosphate, calcium sulfate, calcium carbonate, biological ceramics, bioactive glass, allogeneic bone powder, xenogeneic bone powder, artificial bone powder and biodegradable high polymer repair material bone powder.

A bone hemostatic material with osteogenesis inducing activity comprises active bone extract 0.5-5 wt% of the bone hemostatic material.

Further comprises a dispersing agent accounting for 10 to 23 percent of the bone hemostatic material by mass and a powder auxiliary agent accounting for 72.9 to 89.5 percent of the bone hemostatic material by mass.

Further, the active bone extract comprises the following components in percentage by mass:

blood coagulation agent: 21.3 to 62 percent;

adhesive: 19.5 to 53.7 percent;

a regulator: 2.7-11%;

inducing osteogenesis agent: 6.1-23.4%;

bacteriostatic agent: 1.4-7.5 percent.

Further, the coagulant comprises the following components in percentage by mass of the active bone extract: decorin with molecular weight of 30-60kDa 5.3-21%; 5-14% of prothrombin with molecular weight of 50-90 kDa; 3-8% of coagulation factor with molecular weight of 50-80 kDa; 8-19% of dermatan with molecular weight of 100-doped 360 kDa.

Further, the adhesive comprises the following components in percentage by mass of the active bone extract: 6-18% of keratin with molecular weight of 10-30 kDa; collagen protein with molecular weight of 30-60kDa 8-26%; thrombospondin with molecular weight of 30-130kDa 5.5-9.7%.

Further, the regulator comprises the following components in percentage by mass of the active bone extract: 0.1-1.5% of serum transferrin with molecular weight of 100-160 kDa; 0.1-1.5% of secretory phosphoprotein with molecular weight of 40-60 kDa; 0.6-2.5% of elongation factor with molecular weight of 40-60 kDa; the molecular weight is 400-plus 500kDa heparan sulfate proteoglycan 1.3-3.5%; glucose regulatory protein with molecular weight of 30-50kDa 0.6-2.0%.

Further, the osteogenesis inducing agent comprises the following components in percentage by mass of the active bone extract: ribosomal protein with molecular weight of 40-60kDa 0.5-2.5%; 2.5-10% of a 2-HS-glycoprotein with molecular weight of 80-120 kDa; cartilage protein with molecular weight of 40-60kDa 3-9%; 0.1-1.9% of a bone morphogenetic protein having a molecular weight of 40-60 kDa.

Further, the bacteriostatic agent comprises the following components in percentage by mass: 0.5-2.5% of lysozyme with the molecular weight of 15-35 kDa; tetranectin with a molecular weight of 20-50kDa 0.9-5%.

Application of bone hemostatic material with osteogenesis inducing activity:

when the bone hemostatic material for inducing osteogenesis activity is clinically applied, the bone hemostatic material can be prepared into viscous semisolid, is smeared on a bleeding part of a bone wound surface, can be firmly adhered to the bone wound surface, can effectively stop bleeding, degrades and absorbs at a later stage, releases an induced osteogenesis active substance at the same time, and forms new bones;

or the composition can be pre-filled into an injector, sealed and packaged and then sterilized, so that the composition is clinically used for bone wound hemostasis;

the bone hemostatic material for inducing osteogenesis activity can also be subpackaged into injectors by freeze drying or natural drying, sealed packaging and then sterilized, and a small amount of physiological saline or autologous blood is absorbed when in use and is smeared or injected on the surface of a bone wound for hemostasis.

Compared with the prior art, the bone hemostatic material with the activity of inducing osteogenesis and the preparation method thereof have the following advantages:

the hemostatic material has good adhesion and hemostatic effect, can be degraded, does not cause the problem that the bone wound occupies space or is difficult to repair due to the existence of foreign matters, and has good biological safety and biocompatibility.

The hemostatic effect is good, the bleeding is stopped by mechanical filling in the early stage, the middle-stage hemostatic factor thoroughly prevents the re-bleeding, the hemostatic material is degraded after the hemostasis is finished, the active bone extract with osteoinductive activity enables the blood vessels to grow in, the bone formation of the self body is realized, and the whole process has no foreign body occupation, rejection and other reactions.

The production period is short, the production cost is low, the method is suitable for industrial large-scale production, and the method can be applied to clinical bone wound hemostasis.

Can be randomly plastic, can be smeared, injected and stuffed by doctors according to the condition of the bone wound surface and the plastic according to the requirement, and is convenient to use.

Has good biocompatibility: the whole development process of the biological extraction strictly controls the process parameters, and the strict and comprehensive verification proves that the biological extraction has no immunogenicity, thorough virus inactivation effect and no pyrogen, can achieve the sterile level of implanted medical instruments through sterilization, has no cytotoxicity and has no irritation reaction.

The material is convenient for clinical use, can be prepared into solid form, semisolid form and slurry substance according to the requirement, can be clinically selected according to the requirement, and can also be smeared, adhered and injected on related parts or apparatuses to achieve the aim of clinical treatment.

The material has the unique characteristics that the material can be degraded after hemostasis is finished, new bone generation can be induced in the degradation process, active substances extracted from the bovine bone contain the function of inducing osteoblast generation, and the active substances are released in the degradation process of the material for hemostasis so as to induce osteogenesis and form new bone, so that the material is not occupied and the bone healing is accelerated.

Detailed Description

It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

The invention will be described in detail with reference to the following examples.

Example 1

(1) crushing: crushing the cortical bone blocks by a crusher, wherein liquid nitrogen is used for keeping the temperature low in the crushing process. The cortical bone blocks are crushed into cortical bone granules of 100 meshes to 6 meshes.

(2) Degreasing: the degreasing solution is a mixed solution of trichloromethane and methanol with the volume ratio of 1:1, methanol which is 0.1 time of the weight of cortical bone particles and trichloromethane which is 0.2 time of the weight of the cortical bone particles are sequentially added into a reaction tank, the mixture reacts for 12 hours at room temperature, the degreasing solution is replaced, and the degreasing is carried out again.

(3) Decalcification: preparing decalcifying liquid (0.6mol/L hydrochloric acid), and soaking the cortical bone particles in the decalcifying liquid, wherein the decalcifying liquid just submerges the cortical bone particles. The reaction was carried out at room temperature for 26 hours. The decalcification solution is replaced and decalcification is carried out twice.

(4) Deproteinization: soaking the cortical bone particles in 4mol/L calcium chloride solution, and reacting at room temperature for at least 8h, wherein the calcium chloride solution is over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 0.5 mol/LEDTA.2Na solution, and reacting for 10h at room temperature, wherein the EDTA.2Na solution is required to be over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 7mol/L lithium chloride solution, and reacting for 12h at room temperature if the lithium chloride solution is over the cortical bone particles. Taking out the cortical bone grains, soaking the cortical bone grains in water until the water submerges the cortical bone grains, and reacting for 5h at 60 ℃.

(5) Extraction: extracting the cortical bone particles by using the extracting solution. The extraction liquid is: adding (5mol/L) urea solution, (0.5mol/L) calcium chloride and (0.01mol/L) NEMI into purified water, stirring thoroughly until dissolving, diluting to 1L, adding cortical bone particles into the extract, and reacting at room temperature for 16h to obtain the extract. Concentrating the extractive solution with ultrafilter. The concentrated extract was poured into a container with a semipermeable membrane containing purified water and extract at room temperature for 60 h.

(6) Centrifugal drying: centrifuging the dialyzed extract at 17000RPM for 40min, dispersing the obtained precipitate with water, and placing in a container with a semipermeable membrane with buffer solution of 0.2mol/L citric acid/0.05 mol/L sodium citrate as the bone crude extract while dialyzing at room temperature for 20 hr. After dialysis, centrifugation was carried out again at 40000RPM for 30 min.

(7) And (3) physically mixing the centrifuged precipitate according to the proportion in the table 1, uniformly stirring by using an emulsion stirrer, filling into a pre-filled syringe, sealing in a bubble cap, a plastic suction box or other breathable sealed packages, sterilizing by using ethylene oxide, and resolving to be qualified to obtain the sterile hemostatic material.

The components of the active bone extract and the bone hemostatic material obtained in example 1 are shown in tables 1 and 2 below through mass spectrometry.

Example 2

(2) Degreasing: the degreasing solution is a mixed solution of trichloromethane and methanol with the volume ratio of 1:1, methanol which is 0.3 time of the weight of cortical bone particles and trichloromethane which is 0.7 time of the weight of the cortical bone particles are sequentially added into a reaction tank, the mixture reacts for 16 hours at room temperature, the degreasing solution is replaced, and the degreasing is carried out again.

(3) Decalcification: preparing decalcifying liquid (0.5mol/L hydrochloric acid), and soaking the cortical bone particles in the decalcifying liquid, wherein the decalcifying liquid just submerges the cortical bone particles. The reaction was carried out at room temperature for 28 hours. The decalcification solution is replaced and decalcification is carried out twice.

(4) Deproteinization: soaking the cortical bone particles in 3mol/L calcium chloride solution, and reacting at room temperature for at least 8h, wherein the calcium chloride solution is over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 0.6 mol/LEDTA.2Na solution, and reacting for 8h at room temperature, wherein the EDTA.2Na solution is required to be over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 8mol/L lithium chloride solution, and reacting for 8 hours at room temperature, wherein the lithium chloride solution is far from the cortical bone particles. Taking out the cortical bone grains, soaking the cortical bone grains in water until the water submerges the cortical bone grains, and reacting for 5h at 50 ℃.

(5) Extraction: extracting the cortical bone particles by using the extracting solution. The extraction liquid is: adding (6mol/L) urea solution, (0.6mol/L) calcium chloride, (0.006mol/L) NEMI into purified water, stirring thoroughly until dissolved, diluting to 1L, adding cortical bone particles into the extract, and reacting at room temperature for 18h to obtain the extract. Concentrating the extractive solution with ultrafilter. The concentrated extract was poured into a container partitioned with a semipermeable membrane containing purified water and extract at the same time, and dialyzed at room temperature for 50 h.

(6) Centrifugal drying: centrifuging the dialyzed extract at 18000RPM for 35min, dispersing the obtained precipitate with water again, placing in a container separated by a semipermeable membrane containing 0.2mol/L citric acid/0.05 mol/L sodium citrate buffer solution as bone crude extract dispersion, and dialyzing at room temperature for 20 hr. After dialysis, centrifugation was performed again at 35000RPM for 30 min.

(7) And (3) physically mixing the centrifuged precipitate according to the proportion in the table 1, and repeatedly extruding by using a kneader or a roller press to obtain the pasty semisolid. Sealing the semi-solid in an all-plastic bubble cap, sealing in a Tyvek bag again, sterilizing with ethylene oxide, and resolving to obtain the bone wound hemostatic material with any plasticity and spreadability.

The components of the active bone extract and the bone hemostatic material obtained in example 2 are shown in tables 1 and 2 below through mass spectrometry.

Example 3

(2) Degreasing: the degreasing solution is a mixed solution of trichloromethane and methanol with the volume ratio of 1:1, methanol which is 0.5 time of the weight of cortical bone particles and trichloromethane which is 1.5 times of the weight of the cortical bone particles are sequentially added into a reaction tank, the mixture reacts for 18 hours at room temperature, the degreasing solution is replaced, and the degreasing is carried out again.

(3) Decalcification: preparing decalcifying liquid (0.4mol/L hydrochloric acid), and soaking the cortical bone particles in the decalcifying liquid, wherein the decalcifying liquid just submerges the cortical bone particles. The reaction was carried out at room temperature for 28 hours. The decalcification solution is replaced and decalcification is carried out twice.

(4) Deproteinization: soaking the cortical bone particles in 2mol/L calcium chloride solution, and reacting at room temperature for at least 8h, wherein the calcium chloride solution is over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 0.4 mol/LEDTA.2Na solution, and reacting for 16h at room temperature, wherein the EDTA.2Na solution is just over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 9mol/L lithium chloride solution, and reacting for 16h at room temperature, wherein the lithium chloride solution is far from the cortical bone particles. Taking out the cortical bone grains, soaking the cortical bone grains in water until the water submerges the cortical bone grains, and reacting for 5h at 55 ℃.

(5) Extraction: extracting the cortical bone particles by using the extracting solution. The extraction liquid is: adding (4mol/L) urea solution, (0.8mol/L) calcium chloride and (0.015mol/L) NEMI into purified water, fully stirring until the solution is dissolved, diluting to 1L, adding cortical bone particles into the extract, and reacting at room temperature for 16h to obtain the extract. Concentrating the extractive solution with ultrafilter. The concentrated extract was poured into a container with a semi-permeable membrane into which the purified water was put and the extract was put, and dialyzed at room temperature for 58 hours.

(6) Centrifugal drying: centrifuging the dialyzed extract at 20000RPM for 30min, dispersing the obtained precipitate with water, and placing in a container with a semipermeable membrane containing 0.2mol/L citric acid/0.05 mol/L sodium citrate buffer solution as bone crude extract dispersion for 20 hr at room temperature. After dialysis, centrifugation was carried out again at 30000RPM for 30 min.

(7) Mixing the centrifuged precipitate with dispersant (soybean oil) according to the proportion in table 1, standing overnight, then putting into an emulsion mixer to mix with the powder auxiliary agents (calcium phosphate and bioactive glass) in the proportion in the table, mixing to obtain a paste-shaped hemostatic material, filling into a pre-filled syringe, sealing in a Tyvek bag, sterilizing with ethylene oxide, and resolving to obtain the hemostatic material.

The components of the active bone extract and the bone hemostatic material obtained in example 3 are shown in tables 1 and 2 below through mass spectrometry.

Example 4

(2) Degreasing: the degreasing solution is a mixed solution of trichloromethane and methanol with the volume ratio of 1:1, methanol which is 0.4 time of the weight of cortical bone particles and trichloromethane which is 1.4 times of the weight of the cortical bone particles are sequentially added into a reaction tank, the mixture reacts for 24 hours at room temperature, the degreasing solution is replaced, and the degreasing is carried out again.

(3) Decalcification: preparing decalcifying liquid (0.6mol/L hydrochloric acid), and soaking the cortical bone particles in the decalcifying liquid, wherein the decalcifying liquid just submerges the cortical bone particles. The reaction was carried out at room temperature for 10 hours. The decalcification solution is replaced and decalcification is carried out twice.

(4) Deproteinization:

soaking the cortical bone particles in 4mol/L calcium chloride solution, and reacting at room temperature for at least 8h, wherein the calcium chloride solution is over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 0.8 mol/LEDTA.2Na solution, and reacting for 10h at room temperature, wherein the EDTA.2Na solution is required to be over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 6mol/L lithium chloride solution, and reacting for 30h at room temperature if the lithium chloride solution is over the cortical bone particles. Taking out the cortical bone grains, soaking the cortical bone grains in water until the water submerges the cortical bone grains, and reacting for 5h at 50 ℃.

(5) Extraction: extracting the cortical bone particles by using the extracting solution. The extraction liquid is: adding (7mol/L) urea solution, (0.7mol/L) calcium chloride and (0.02mol/L) NEMI into purified water, stirring thoroughly until dissolving, diluting to 1L, adding cortical bone particles into the extract, and reacting at room temperature for 16h to obtain the extract. Concentrating the extractive solution with ultrafilter. The concentrated extract was poured into a container with a semipermeable membrane containing purified water and extract at room temperature for 60 h.

(6) Centrifugal drying: centrifuging the dialyzed extract at 19000RPM for 40min, dispersing the obtained precipitate with water again, placing in a container with a semipermeable membrane, separating with 0.2mol/L citric acid/0.05 mol/L sodium citrate buffer solution as bone crude extract dispersion, and dialyzing at room temperature for 20 hr. After dialysis, centrifugation was carried out again at 30000RPM for 30 min.

(7) Mixing the centrifuged precipitate with dispersing agents (soybean lecithin 5, vitamin E5 and soybean lecithin 5) according to the proportion in the following table 1, standing overnight, then putting into an emulsion mixer to mix with powder auxiliary agents (calcium sulfate 70 and calcium carbonate 14) according to the proportion in the following table, obtaining a viscous pasty hemostatic material after mixing, filling into a pre-filled and sealed injector, sealing in a Tyvek bag, performing ethylene oxide sterilization, and obtaining the hemostatic material after analysis.

The components of the active bone extract and the bone hemostatic material obtained in example 4 are shown in tables 1 and 2 below through mass spectrometry.

Example 5

(2) Degreasing: the degreasing solution is a mixed solution of trichloromethane and methanol with the volume ratio of 1:1, methanol which is 0.6 times of the weight of cortical bone particles and trichloromethane which is 2 times of the weight of the cortical bone particles are sequentially added into a reaction tank, the mixture reacts for 15 hours at room temperature, the degreasing solution is replaced, and the degreasing is carried out again.

(3) Decalcification: preparing decalcifying liquid (0.6mol/L hydrochloric acid), and soaking the cortical bone particles in the decalcifying liquid, wherein the decalcifying liquid just submerges the cortical bone particles. The reaction was carried out at room temperature for 30 hours. The decalcification solution is replaced and decalcification is carried out twice.

(4) Deproteinization: soaking the cortical bone particles in 3mol/L calcium chloride solution, and reacting at room temperature for at least 8h, wherein the calcium chloride solution is over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 0.7 mol/LEDTA.2Na solution, and reacting for 10h at room temperature, wherein the EDTA.2Na solution is required to be over the cortical bone particles. Taking out the cortical bone particles, soaking the cortical bone particles in 10mol/L lithium chloride solution, and reacting for 12h at room temperature if the lithium chloride solution is over the cortical bone particles. Taking out the cortical bone grains, soaking the cortical bone grains in water until the water submerges the cortical bone grains, and reacting for 5h at 60 ℃.

(5) Extraction: extracting the cortical bone particles by using the extracting solution. The extraction liquid is: adding (8mol/L) urea solution, (0.4mol/L) calcium chloride, and (0.018mol/L) NEMI into purified water, stirring thoroughly until dissolved, diluting to 1L, adding cortical bone particles into the extract, and reacting at room temperature for 16h to obtain the extract. Concentrating the extractive solution with ultrafilter. The concentrated extract was poured into a container with a semipermeable membrane containing purified water and extract at room temperature for 60 h.

(6) Centrifugal drying: centrifuging the dialyzed extract at 15000RPM for 40min, dispersing the obtained precipitate with water again, placing in a container with a semipermeable membrane with buffer solution of 0.2mol/L citric acid/0.05 mol/L sodium citrate as the bone crude extract while separating, and dialyzing at room temperature for 20 hr. After dialysis, centrifugation was carried out again at 40000RPM for 30 min.

(7) Mixing the centrifuged precipitate with dispersing agents (soybean lecithin 15 and vitamin E5) according to the proportion in the following table 1, standing overnight, then putting into an emulsion mixer to be mixed with powder auxiliary agents (hydroxyapatite 70 and calcium phosphate 6) according to the proportion in the following table, obtaining a viscous pasty hemostatic material after mixing, filling into a pre-filled syringe, sealing in a Tyvek bag, performing ethylene oxide sterilization, and obtaining the hemostatic material after analysis.

The components of the active bone extract and the bone hemostatic material obtained in example 5 are shown in tables 1 and 2 below through mass spectrometry.

Comparative example 1

A bone hemostatic material having osteogenesis inducing activity was prepared in substantially the same manner as in example 1, except that:

the extraction procedure of comparative example 1 has no temperature requirement for pulverization, and the temperature is not maintained at-20 ℃ and below, compared to example 1; the reaction time of degreasing and decalcification is longer, the centrifugation time is longer, and in the step of removing hybrid protein, cortical bone grains are sequentially soaked in 5mol/L lithium chloride solution and water; the extraction liquid in the extraction step has no urea, and the dialysis time at room temperature is longer.

The components of the active bone extract and the bone hemostatic material obtained in comparative example 1 are shown in tables 1 and 2 below through mass spectrometry.

Comparative example 2

the extraction procedure of comparative example 2 has no temperature requirement for pulverization and the temperature is not maintained at-20 ℃ and below, compared to example 1; in the step of removing hybrid protein, the cortical bone particles are sequentially soaked in EDTA-2 Na solution and water; the extraction liquid in the extraction step is free of calcium chloride, and the dialysis time at room temperature is longer.

The components of the osteoinductive active extract obtained in comparative example 2 are shown in tables 1 and 2 below through mass spectrometry.

Comparative example 3

the extraction procedure of comparative example 3 has no temperature requirement for pulverization and the temperature is not maintained at-20 ℃ and below, compared to example 1; the reaction time of degreasing and decalcification is longer, the centrifugation time is longer, and in the step of removing hybrid protein, the cortical bone particles are sequentially soaked in calcium chloride solution and water; the extraction liquid in the extraction step has no N-ethylmaleimide, and the dialysis time at room temperature is longer.

The components of the active bone extract and the bone hemostatic material obtained in comparative example 3 are shown in tables 1 and 2 below through mass spectrometry.

Table 1 active bone extract ingredient ratio table of examples and comparative examples

TABLE 2 list of component ratios of bone hemostatic materials of examples and comparative examples

As can be seen from the above table, the active bone extracts obtained in example 1 and comparative examples 1-3 differ in their components:

the active bone extract obtained in example 1 contains 17.5% decorin and 14% dermatopontin;

the active bone extract obtained in comparative example 1 had no decorin, and 31.5% dermatopontin, and the ratio of dermatopontin was the same as the total ratio of decorin and dermatopontin in example 1, so that the total ratio of the coagulant in the active bone extract was not changed.

The active bone extract obtained in the comparative example 2 has the decorin content of 31.5% and no adiponectin; and the ratio of the dermatopontin to the total amount of the decorin and dermatopontin components in example 1 was the same, so that the total ratio of the coagulant to the active bone extract was not changed.

The active bone extract obtained in comparative example 3 had neither decorin nor dermatopontin. However, the relative content of prothrombin and coagulation factor in the blood coagulation agent is not changed, and the total ratio of the blood coagulation agent in the active bone extract is not changed.

The active bone extract of the embodiment 1 contains 2 components of decorin and dermatopontin, and the 2 components have synergistic effect, so that the hemostasis time of the prepared hemostatic material is fully shortened, and the secondary bleeding phenomenon is avoided; in addition, the hemostatic material can have bone induction activity, so that blood vessels can grow in, autogenous bone formation can be realized, and the induced bone formation activity is obviously improved.

Compared with comparative examples 1-3, the preparation method in the examples has shorter use time, adopts low-temperature crushing, and is beneficial to the expression of protein activity; the urea solution, the calcium chloride and the NEMI are added into the extract, so that 2 components of decorin and dermatopontin are added in the finally obtained active bone extract, the synergistic effect of the 2 components is realized, the hemostasis time of the prepared hemostatic material is fully shortened, and the secondary bleeding phenomenon is avoided; in addition, the hemostatic material can have bone induction activity, so that the hemostatic material can grow blood vessels, form autologous bone and obviously improve the bone induction activity

The excellent performances of the product of the invention, such as safety, effectiveness, stability, biocompatibility, induced osteogenesis activity, and the like, can be proved by the following series of experiments.

1. Safety feature

1.1 verification of Virus inactivation

To verify the safety of this material, we performed virus inactivation verification tests on multiple batches of product.

The test mechanism comprises: china institute for testing biological products

And (3) indicating virus: simian vacuolating virus 40, pseudorabies virus, vesicular stomatitis virus and reovirus type III

And (5) verifying and concluding: after verification, the bone hemostatic material with the activity of inducing osteogenesis is prepared according to the production process after infecting simian vacuolating virus 40, pseudorabies virus, vesicular stomatitis virus and reovirus III type indicator virus, and the inactivation amount of the indicator virus is more than 6 logs. Therefore, the production process of the material can play an effective virus inactivation role.

1.2 cytotoxicity

The cytotoxicity test is a toxicological risk assessment using in vitro cell culture methods. The test was carried out according to the national standard GB/T16886.5 "part 5 of the biological evaluation of medical instruments: the MTT method is adopted for detection by an extract method in vitro cytotoxicity test.

The specific test steps are as follows: the samples of examples 1 to 5 were diluted with a serum-containing medium containing 10% at a ratio of 0.1g/mL to prepare test solutions. The sample test solution, negative control (polyethylene material), positive control (5% DMSO) extract and medium control (serum-containing medium) were placed in wells of 5L 929 mouse fibroblast cell culture plates containing 5% CO at 37 deg.C₂The culture box of (2) was cultured, and after 72 hours, the cells cultured in the sample test group, the negative control group, the positive control group and the medium control group were observed under a microscope, and the relative proliferation rate was measured by the MTT method.

The cytotoxicity response ratings are based on specific values of relative proliferation rates, as shown in table 3 below:

TABLE 3 grading of cytotoxic reactions

Level of cytotoxicity	Relative proliferation rate%
		0	≥100
1	80-99
		2	50-79
3	30-49
		4	0-29

The higher the relative proliferation rate, the lower the cytotoxicity, demonstrating better safety of the material.

TABLE 4 cytotoxicity test results tabulated

From the test results in the table above, it can be seen that the hemostatic material of the present invention and the hemostatic material of the comparative example have higher cell proliferation rate, and the in vitro cytotoxicity test level is not greater than 1, which fully indicates that the hemostatic material has no cytotoxicity and higher safety.

The hemostatic material of the invention and the hemostatic material of the comparative example are both biologically extracted, so the safety is high.

1.3 blood compatibility

The blood compatibility test is an evaluation of the desirable response of blood to exogenous substances or materials. Generally refers to the compatibility of the material with the blood components. The test is carried out according to the 4 th point of the national standard GB/T16886.4-2003 medical instrument biological evaluation: the blood interaction test selection is carried out, and the standard requires that the hemolysis rate is lower than 5 percent, which indicates that the test article has good compatibility with blood.

The specific test steps are as follows: samples of examples 1-5 and comparative examples 1-3 were extracted at 37. + -. 1 ℃ for 72. + -.2 h, and the extraction medium was normal saline. The specific test steps are as follows: the samples of examples 1 to 5 were taken as the samples of comparative examples 1 to 3, and the hemolysis test was conducted.

TABLE 5 listing of results of the blood compatibility test

Test article	Hemolysis ratio (%)
		Example 1	0.01
Example 2	0.01
		Example 3	0.02
Example 4	0.01
		Example 5	0.03
Comparative example 1	0.02
		Comparative example 2	0.02
Comparative example 3	0.01

From the experimental results in the above table, it can be seen that compared with the comparative example, the hemostatic material of the present invention and the material of the comparative example both have very low hemolysis rate, which fully indicates that the hemostatic material has good compatibility with blood, no toxicity and high safety.

1.4 pyrogen reaction

The pyrogen test is used for evaluating whether the test article has potential pyrogenicity after being implanted into a human body. The test was carried out according to the national standard GB/T16886.12-2005 "part 12 of the biological evaluation of medical devices: sample preparation and reference sample "to perform the test, wherein the body temperature rise of three rabbits in the initial test is lower than 0.6 ℃, and the total body temperature rise of 3 rabbits is lower than 1.3 ℃; or the total sum of the temperature rise of the initial test and the secondary test combined with 8 rabbits is 3.5 or less than 3.5, and the pyrogen examination of the test sample is judged to be in accordance with the regulation. Among 3 rabbits in the initial test, more than 1 rabbit with a body temperature rise of 0.6 ℃ or above 0.6 ℃; or more than 1 rabbit with the body temperature rising by 0.6 ℃ or more than 0.6 ℃ in 5 retested rabbits; or the total body temperature rise of 8 rabbits in the initial test and the retest exceeds 3.5, and the pyrogen examination of the test article is judged not to meet the regulation.

The specific test steps are as follows: samples of examples 1-5 and comparative examples 1-3 were extracted at 37 + -1 deg.C for 72 + -2 h with normal saline as the extraction medium, and the extract was subjected to pyrogen examination. The sum of the temperature increases of the three animals was recorded at the end of the experiment.

TABLE 6 list of pyrogen reaction test results

Test article	Pyrogen reaction
		Example 1	0
Example 2	0.1
		Example 3	0.1
Example 4	0.6
		Example 5	0.1
Comparative example 1	0.5
		Comparative example 2	0.7
Comparative example 3	0.3

As can be seen from the experimental results in the table above, the hemostatic material of the present invention and the comparative material have very low pyrogen reaction, which fully indicates that the hemostatic material has no potential pyrogenicity and high safety after being implanted into human body.

2. Effectiveness of

2.1. Ilium test in rabbits

The ilium cancellous bone has a large area, rich bone pore network structures and large pore diameters, is the site of the organism with the most bleeding from the skeletal bone wound surface, and has a representative hemostatic effect when the bone hemostatic material is observed at the site.

Healthy New Zealand rabbits are selected, and the male and female rabbits are unlimited and have the weight of 2.5-3.0 Kg. 16 experimental animals were taken, anesthetized by injection, and the operated animals were positioned on the operating table in the prone position. The hair removal treatment is performed in the operation area and the skin is disinfected. And respectively making lateral incisions on two sides of iliac crest, stripping periosteum, leaking an external iliac plate, drilling a wound surface with the width of about 1cm by using a bone drill, stopping bleeding by using the samples of the embodiment and the comparative example of the invention after the wound surface bleeds, observing the hemostatic effect, observing whether the sample can be firmly adhered to the bone wound surface, continuously observing for 30min, and observing whether new blood oozing occurs in the time period.

TABLE 7 Rabbit ilium test results List

The above table shows that the hemostatic material of the invention has good coating adhesion, arbitrary plasticity, convenient coating, firm adhesion to the bone wound surface, good hemostatic effect, and immediate hemostasis, and no new blood seepage occurs within 30 min. The hemostatic materials in the comparative examples have slower hemostasis speed and are less effective than those in the examples, and the bleeding phenomenon occurs.

2.2 Canine radius test

The canine radius was prepared as a lesion of bone defect, and the wound surface was filled with example 1 and comparative example 1, respectively. The oozing of blood is quickly stopped, and the focus is closed. After 6 months of operation, the implanted group is inspected by a general specimen, and the implanted group in example 1 obviously shows that the new bone tissues repair the bone defects. After 6 months of surgery, the example 1 implant group new bone bridges the bone defect at both ends, and little cartilage formation is still visible in the prosthetic new bone. In contrast, the comparative example 1 group had less new bone tissue and the post-operative bridging of both ends of the six-month bone defect was less complete than in the example 1 group.

As seen from the above safety test results and effectiveness test results, although the hemostatic materials of the comparative example and the hemostatic materials of the present invention have high safety, the effectiveness of the hemostatic materials of the comparative example is far less than that of the hemostatic materials of the present invention. The material disclosed by the invention has good induced osteogenesis activity, good adhesion and good hemostatic effect, can be degraded, and can not cause the problems of bone wound occupation or difficult repair of the bone wound due to the existence of foreign matters.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the invention, so that any modifications, equivalents, improvements and the like, which are within the spirit and principle of the present invention, should be included in the scope of the present invention.

Claims

1. A method for preparing a bone hemostatic material having an osteogenesis-inducing activity, comprising: the method comprises the following steps:

2. The bone hemostatic material with osteogenesis-inducing activity, which is prepared by the method for preparing a bone hemostatic material with osteogenesis-inducing activity according to claim 1, wherein: comprises active bone extract accounting for 0.5-5% of the bone hemostatic material by mass.

3. The bone hemostatic material having osteogenesis inducing activity of claim 2, wherein: also comprises a dispersant accounting for 10 to 23 percent of the bone hemostatic material by mass and a powder auxiliary agent accounting for 72.9 to 89.5 percent of the bone hemostatic material by mass.

4. A bone hemostatic material with osteogenesis inducing activity according to claim 2 or 3, characterized by: the active bone extract comprises the following components in percentage by mass:

blood coagulation agent: 21.3 to 62 percent;

adhesive: 19.5 to 53.7 percent;

a regulator: 2.7-11%;

inducing osteogenesis agent: 6.1-23.4%;

bacteriostatic agent: 1.4-7.5 percent.

5. A bone hemostatic material having osteogenesis inducing activity according to claim 4, wherein: the coagulant comprises the following components in percentage by mass: decorin with molecular weight of 30-60kDa 5.3-21%; 5-14% of prothrombin with molecular weight of 50-90 kDa; 3-8% of coagulation factor with molecular weight of 50-80 kDa; 8-19% of dermatan with molecular weight of 100-doped 360 kDa.

6. A bone hemostatic material having osteogenesis inducing activity according to claim 4, wherein: the adhesive comprises the following components in percentage by mass of the active bone extract: 6-18% of keratin with molecular weight of 10-30 kDa; collagen protein with molecular weight of 30-60kDa 8-26%; thrombospondin with molecular weight of 30-130kDa 5.5-9.7%.

7. A bone hemostatic material having osteogenesis inducing activity according to claim 4, wherein: the regulator comprises the following components in percentage by mass of the active bone extract: 0.1-1.5% of serum transferrin with molecular weight of 100-160 kDa; 0.1-1.5% of secretory phosphoprotein with molecular weight of 40-60 kDa; 0.6-2.5% of elongation factor with molecular weight of 40-60 kDa; the molecular weight is 400-plus 500kDa heparan sulfate proteoglycan 1.3-3.5%; glucose regulatory protein with molecular weight of 30-50kDa 0.6-2.0%.

8. A bone hemostatic material having osteogenesis inducing activity according to claim 4, wherein: the induced osteogenesis agent comprises the following components in percentage by mass of the active bone extract: ribosomal protein with molecular weight of 40-60kDa 0.5-2.5%; 2.5-10% of a 2-HS-glycoprotein with molecular weight of 80-120 kDa; cartilage protein with molecular weight of 40-60kDa 3-9%; 0.1-1.9% of a bone morphogenetic protein having a molecular weight of 40-60 kDa.

9. A bone hemostatic material having osteogenesis inducing activity according to claim 4, wherein: the bacteriostatic agent comprises the following components in percentage by mass: 0.5-2.5% of lysozyme with the molecular weight of 15-35 kDa; tetranectin with a molecular weight of 20-50kDa 0.9-5%.