CN1200728C - Injection-type bone morphogenetic protein using fibrin as carrier - Google Patents

Abstract

The invention discloses an injection-type bone morphogenetic protein with fibrin as a carrier, which is suitable for the treatment of clinical fractures, nonunions and bone defects. It is based on the fibrin isolated from the blood of patients or healthy blood donors as a carrier, and the compound bovine bone morphogenetic protein is directly injected percutaneously into the injured part to exert the slow-release effect of the carrier and the bone morphogenetic protein to promote new bone growth and Fracture healing and accelerated wound repair. The invention has no immunogenicity and rejection reaction, and no adverse reaction after being implanted in the body. Fibrin is in a liquid state before use, and can be rapidly solidified in the body and can be shaped arbitrarily. At the same time, the hemostatic and adhesive effects of fibrin are beneficial to prevent the formation of local hematoma and provide a good local environment for bone regeneration and healing. The present invention has the advantages of definite therapeutic effect, wide application range, simple and convenient operation, safety and reliability confirmed by animal experiment and clinical experiment.

Description

Injectable Bone Morphogenetic Protein with Fibrin as Carrier

1. Field

The invention relates to a protein injection in the medical field, in particular to a bone morphogenetic protein that can be used for injection in the treatment of clinical fractures, nonunions, bone defects and other diseases.

2. Background technology

The main role of bone morphogenetic proteins is to promote the recruitment and differentiation of undifferentiated mesenchymal and osteocyte cells into osteoblasts. Because of its high-efficiency osteoinductive activity, it has excellent clinical application prospects in promoting fracture healing and treating bone defects and nonunions. Most of the existing clinical methods are compounding bone morphogenetic proteins on solid materials and implanting them into traumatic sites through surgery. The operation is complicated, the cost is high, and the scope of application is limited, especially for closed fractures and other unsightly fractures that occur most frequently in clinical practice. Cases requiring surgical treatment are difficult to apply.

3. Contents of the invention

According to the partial defects or deficiencies in the above-mentioned prior art, the purpose of the present invention is to provide a nonunion and bone defect patients who need surgical treatment, and can also use ordinary syringes for percutaneous direct injection, so it can be used for all patients with Patients with bone injuries. The injection-type bone morphogenetic protein with human fibrin as the carrier has the advantages of wide application range, simple operation and good therapeutic effect.

In order to achieve the above-mentioned purpose, the technical concept adopted in the present invention is to use the blood of patients or healthy adults in blood banks to quickly prepare fibrinogen as the carrier of bone morphogenetic protein by low-temperature centrifugation, and use thrombin and aprotinin Acts to convert the fibrinogen/bone morphogenetic protein complex into a fibrin clot after injection into the body. The slow absorption of fibrin clot by the body is used to slowly release bone morphogenetic protein, so that bone morphogenetic protein can play its biological role in the whole process of wound repair, and achieve the effect of inducing new bone formation and promoting wound repair .

The technical solutions adopted are:

The injection-type bone morphogenetic protein with fibrin as the carrier is prepared according to the following steps:

1) Obtain healthy adult anticoagulant blood from the hospital blood bank/blood bank or aseptically collect the patient's autologous venous blood before treatment;

2) Centrifuge at 3000RPM for 20 minutes to separate plasma, extract fibrinogen by refrigerated centrifugation, add bovine bone morphogenetic protein extract, the ratio is (2-5) mg: 1 mg (fibrinogen: bone morphogenetic protein); the above mixture Freeze-dry at -40°C to make flocs for later use;

3) Prepare the fibrinogen/bone morphogenetic protein mixture with physiological saline to form a suspension with a concentration of 4mg/ml to 8mg/ml as component A; prepare a calcium chloride solution with a concentration of 250IU/ml to 400IU/ml for blood coagulation The enzyme solution is component B;

4) Add aprotinin at a concentration of 0.5 mg/ml to 50 mg/ml to component B.

Another feature of the present invention is that the bovine bone morphogenetic protein extract is a mixture of various bone morphogenetic proteins artificially extracted from fresh calf cortical bone according to conventional methods. The main steps are degreasing, deproteinizing, and guanidine hydrochloride , Extracted with urea, dried at room temperature, proved to have osteoinductive activity by mouse muscle bag experiment.

The invention utilizes the dissolving properties of fibrinogen to make injections, so it can not only be used for nonunion and bone defect patients who need surgical treatment, but also can be injected directly through the skin with ordinary syringes, so it can be used for all patients with bone injuries . When in use, add equal volumes of component A and component B into the two barrels of the double-barreled syringe, and mix well during the injection process. The invention has the advantages of wide application range, simple and convenient operation, definite therapeutic effect and the like. Fibrin is extracted from human blood and then used in the human body. There is no immunogenicity and rejection, and there is no adverse reaction after implantation in the body. In particular, drawing the patient's own blood before treatment can also avoid the spread of blood diseases. Because only the plasma components in the blood are used, other components can be reinfused back to the patient after treatment, so there will be no adverse effects on the patient's blood system. Fibrin is in a liquid state before use, and can be rapidly solidified in the body and can be shaped arbitrarily. At the same time, the hemostatic and adhesive effects of fibrin are beneficial to prevent the formation of local hematoma and provide a good local environment for bone regeneration and healing.

4. Specific implementation

Below in conjunction with embodiment the present invention is described in further detail.

Embodiment 1: According to the technical scheme of the present invention, the injection-type bone morphogenetic protein with fibrin as the carrier is prepared according to the following steps:

1) Obtain healthy adult anticoagulant blood from the hospital blood bank/blood bank or aseptically collect the patient's autologous venous blood before treatment;

2) Centrifuge at 3000RPM for 20 minutes to separate plasma, extract fibrinogen by refrigerated centrifugation, add bovine bone morphogenetic protein extract at a ratio of 2mg: 1mg (fibrinogen: bone morphogenetic protein); freeze-dry the above mixture at -40°C Make flocs for later use;

Bovine bone morphogenetic protein extract is a mixture of various bone morphogenetic proteins artificially extracted from fresh calf cortical bone according to conventional methods. Mouse muscle bag experiment confirmed that it has osteoinductive activity;

3) The fibrinogen/bone morphogenetic protein mixture is formulated with physiological saline to form a suspension with a concentration of 8mg/ml as component A; the calcium chloride solution is used to prepare a thrombin solution with a concentration of 250IU/ml as component B;

4) Add aprotinin at a concentration of 50 mg/ml to component B.

Embodiment 2: According to the technical scheme of the present invention, the injection-type bone morphogenetic protein with fibrin as the carrier is prepared according to the following steps:

1) Obtain healthy adult anticoagulant blood from the hospital blood bank/blood bank or aseptically collect the patient's autologous venous blood before treatment;

2) Centrifuge at 3000RPM for 20 minutes to separate plasma, extract fibrinogen by refrigerated centrifugation, add bovine bone morphogenetic protein extract at a ratio of 4mg: 1mg (fibrinogen: bone morphogenetic protein); freeze-dry the above mixture at -40°C Make flocs for later use;

Bovine bone morphogenetic protein extract is a mixture of various bone morphogenetic proteins artificially extracted from fresh calf cortical bone according to conventional methods. Mouse muscle bag experiment confirmed that it has osteoinductive activity;

3) The fibrinogen/bone morphogenetic protein mixture was formulated with physiological saline to form a suspension with a concentration of 4 mg/ml as component A; the calcium chloride solution was used to prepare a thrombin solution with a concentration of 300 IU/ml as component B;

4) Add aprotinin at a concentration of 30 mg/ml to component B.

Embodiment 3: According to the technical scheme of the present invention, the injection-type bone morphogenetic protein with fibrin as the carrier is prepared according to the following steps:

1) Obtain healthy adult anticoagulant blood from the hospital blood bank/blood bank or aseptically collect the patient's autologous venous blood before treatment;

2) Centrifuge at 3000RPM for 20 minutes to separate plasma, extract fibrinogen by refrigerated centrifugation, add bovine bone morphogenetic protein extract at a ratio of 5mg: 1mg (fibrinogen: bone morphogenetic protein); freeze-dry the above mixture at -40°C Make flocs for later use;

Bovine bone morphogenetic protein extract is a mixture of various bone morphogenetic proteins artificially extracted from fresh calf cortical bone according to conventional methods. Mouse muscle bag experiment confirmed that it has osteoinductive activity;

3) The fibrinogen/bone morphogenetic protein mixture is formulated with physiological saline to form a suspension with a concentration of 6 mg/ml as component A; the calcium chloride solution is used to prepare a thrombin solution with a concentration of 400 IU/ml as component B;

4) Add aprotinin at a concentration of 0.5 mg/ml to component B.

Embodiment 4: According to the technical scheme of the present invention, the injection-type bone morphogenetic protein with fibrin as the carrier is prepared according to the following steps:

1) Obtain healthy adult anticoagulant blood from the hospital blood bank/blood bank or aseptically collect the patient's autologous venous blood before treatment;

2) Centrifuge at 3000RPM for 20 minutes to separate plasma, extract fibrinogen by refrigerated centrifugation, add bovine bone morphogenetic protein extract at a ratio of 3mg: 1mg (fibrinogen: bone morphogenetic protein); freeze-dry the above mixture at -40°C Make flocs for later use;

Bovine bone morphogenetic protein extract is a mixture of various bone morphogenetic proteins artificially extracted from fresh calf cortical bone according to conventional methods. Mouse muscle bag experiment confirmed that it has osteoinductive activity;

3) The fibrinogen/bone morphogenetic protein mixture was formulated with physiological saline to form a suspension with a concentration of 6 mg/ml as component A; the calcium chloride solution was used to prepare a thrombin solution with a concentration of 350 IU/ml as component B;

4) Add aprotinin at a concentration of 15 mg/ml to component B.

The preparation concentration of above-mentioned calcium chloride solution can be adjusted between 250IU/ml～400IU/ml, and the concentration of aprotinin added in B is adjusted between 0.5mg/ml～50mg/ml, all can reach the purpose of the present invention.

Specific example 1: The treatment process of patients with fractures and postoperative nonunion with this repair material is as follows: Manipulative reduction, after observing that the alignment of the fracture end is satisfactory under the fluoroscopy of a TV X-ray machine, take 10ml of this material, and inject it percutaneously under fluoroscopy. Broken ends. After 4 weeks of plaster or external fixator fixation, the fracture healed well. It shows that the material can effectively accelerate the healing speed of fractures, and can avoid the second operation for the treatment of bone nonunion.

Specific example 2: patients with vertebral body compression fractures draw venous blood before operation, make fibrin, and compound bone morphogenetic protein for future use. The fracture is reduced or the compressed vertebral body is stretched through surgery, and internal fixation is used. The pre-prepared fibrin/bone morphogenetic protein and component B are mixed and injected into the vertebral body, and the wound is closed by conventional methods. Cellular components of autologous blood can be reinfused back into the patient during surgery to compensate for blood loss during surgery. In such trauma treatment, this material can not only promote fracture healing, but also induce new bone formation inside the vertebral body and restore the original shape and mechanical strength of the vertebral body.

The syringe is specially designed for one-time use. The features are: 1. The two barrels are parallel, each with a volume of 5ml, and the rear ends of the two propellers are connected, and a propulsion handle is used. 2. The channels at the two outlets are spiral and converge at the end, which is convenient for the two components to mix evenly. 3. There are 3 external needles, the thin needle is a 12-gauge steel common needle for percutaneous injection; the thick needle is a special short needle with an inner diameter of 1.8mm for use in surgery; the nozzle is specially made to make the liquid mix evenly before spraying into fog.

An appropriate amount of aprotinin is added to component B to slow down the absorption rate of fibrin.

The scope of application of the present invention includes orthopedic surgery, neurosurgery, oral surgery and the like.

Before use, add equal volumes of component A and component B into the two barrels of the double-barreled syringe, connect a special needle externally, and inject directly into the injured part percutaneously under the guidance of a TV X-ray machine.

The invention has the advantages of promoting fracture healing, accelerating bone formation and definite effect of bone healing; easy to use, wide indications; good tissue compatibility, safe and reliable.

Claims (2)

1. one kind is the preparation method of the injection-type bone morphogenetic protein of carrier with the fibrin, it is characterized in that, carries out according to the following steps:

1) obtains the preceding aseptic patient's of the taking autogenous vein blood of health adult's anticoagulation or treatment from hospital blood bank/blood station;

2) extract Fibrinogen after the separated plasma, the adding mass ratio is that the Bovine Bone Morphoge Protnetin extract of 2～5: 1 dosage is standby;

3) Fibrinogen/Bovine Bone Morphoge Protnetin mixture is mixed with normal saline, the suspension that is made into mixture concentration and is 4mg/ml～8mg/ml is as composition A; With the calcium chloride solution compound concentration is that 250IU/ml～400IU/ml thrombin solution is composition B; Composition A does not mix external with composition B;

4) adding concentration in composition B is the aprotinin of 0.5mg/ml～50mg/ml.

2. according to claim 1 is the preparation method of the injection-type bone morphogenetic protein of carrier with the fibrin, it is characterized in that: described Bovine Bone Morphoge Protnetin extract, it is the artificial multiple bone morphogenetic protein mixture that extracts from the fresh cortical bone of calf, extracting method is: defat, deproteinization, guanidine hydrochloride, urea extraction have bone-inducting active through mice flesh bag experiment confirm.