阅读说明：本技术 具备骨诱导活性的同种骨修复材料及其制法及其用途 (Allogeneic bone repair material with bone induction activity and preparation method and application thereof ) 是由 尹曼莉 王耀宏 张娟 李晶 张顺聪 于 2019-02-19 设计创作，主要内容包括：本发明提供了一种同种骨修复材料。具体地本发明提供了一种纤维状同种骨材料和该同种骨材料的制备方法和使用方法。本发明的同种骨修复材料具备骨诱导活性并保留了大量哈氏系统结构。(The invention provides an allogeneic bone repair material. In particular to a fibrous same bone material and a preparation method and a use method thereof. The allogeneic bone repair material has bone induction activity and reserves a large number of Hastelloy system structures.)

1. The homogeneous bone repair material is characterized by being fibrous homogeneous bone material.

2. The allograft bone repair material of claim 1, wherein the allograft bone material has a hastelloy system structure.

3. The allograft bone repair material of claim 1, wherein the thickness of the allograft bone material is from 0.001 mm to 2 mm.

4. The allograft bone repair material of claim 1, wherein the homogenous bone material has a lay-flat area S of 0.9 to 120mm²。

5. The homogeneous bone repair material of claim 1, wherein the homogeneous bone material has a ratio of area to thickness, S/, of from 3 to 12000.

6. The homogeneous bone repair material according to claim 1, wherein the homogeneous bone material has one or more of the following characteristics:

i) the length range of the same bone material is 0.1-200 mm;

ii) the average length of the homogeneous bone material is 20-100 mm; (ii) a And/or

iii) about 50-90% of the bone material of the same type has a length L of 30-80 mm.

7. The allograft bone repair material of claim 1, wherein the width W of the allograft bone material is in the range of 0.2mm to 10 mm.

8. The method of preparing an allograft bone repair material of claim 1, said method comprising the steps of:

(1) providing a pretreated allogeneic bone;

(2) processing the pretreated allogeneic bone into a fibrous shape to obtain a fibrous allogeneic bone;

(3) performing demineralization treatment on the fibrous allogeneic bone obtained in the step (2) to obtain demineralized allogeneic bone; and

(4) and (3) cleaning and/or drying the demineralized allogeneic bone obtained in the step (3) to obtain the allogeneic bone repair material.

9. The method of claim 8, wherein the pretreated allogeneic bone in step (1) is obtained from a source of allogeneic bone by a pretreatment step comprising:

(a) providing an allogeneic bone raw material:

(b) removing soft tissues of the allogeneic bone raw material to obtain the allogeneic bone with the soft tissues removed; and

(c) and carrying out degreasing treatment and virus inactivation treatment on the allogeneic bone with the soft tissue removed to obtain the pretreated allogeneic bone.

10. A method of using the allograft bone repair material of claim 1, comprising the steps of:

mixing the bone repair material according to claim 1 with a rehydration liquid for rehydration to obtain a rehydrated bone repair material.

Technical Field

The invention relates to the field of medical materials, in particular to an allogeneic bone repair material with bone induction activity, a preparation method and application thereof.

Background

The incidence of bone tissue damage caused by aging of social population, traffic accidents, natural disasters and the like is increased dramatically, and the life health of human beings is seriously harmed. Therefore, the repair of bone defects has become the subject of intensive research in recent years in the medical community.

Autologous bone grafting is called the "gold standard" for bone grafting because it does not cause rejection and can be perfectly fused with host tissues, but it also has significant disadvantages. The autologous bone graft is generally taken from the ilium of a patient, has limited sources, and is easy to cause complications and increase the pain of the patient.

The transplantation of the allogeneic bone has a long history, and a large number of clinical tests prove that the success rate of the transplantation of the allogeneic bone is close to that of the autologous bone. With the massive application of allogeneic bones, more and more varieties and process preparations are continuously perfected and innovated.

Demineralized Bone Matrix (DBM) is a composite natural bone graft material composed of collagen, non-collagen, and growth factors at low concentration. In 1965, Urist first found a substance capable of inducing bone formation in DBM and defined it as Bone Morphogenetic Proteins (BMPs). Research shows that the DBM implant is an effective and ideal bone repair scaffold material.

Because it is derived from fresh allogeneic bone and is easy to cause immune rejection, the primary reason for the failure of transplantation is that antigen reduction treatment is required. However, the antigenicity of the decalcified bone matrix has a common material basis with the osteoinductivity, and the induced osteogenic material is destroyed while the antigenicity is eliminated.

At present, DBM materials are in powder or flake form, and the using effect is not satisfactory.

In view of the foregoing, there is an urgent need in the art to develop a novel bone repair material having both excellent bone repair properties and low or no immunogenicity.

Disclosure of Invention

The present invention is directed to a novel bone repair material having both excellent bone repair properties and low or no immunogenicity.

In a first aspect of the invention, an allograft repair material is provided which is a fibrous allograft material.

In another preferred example, the allograft bone repair material is a ribbon allograft bone material.

In another preferred embodiment, the homogeneous bone material has a hastelloy system structure.

In another preferred embodiment, the thickness of the same bone material is 0.001-2 mm.

In another preferred example, the thickness of the same bone material is 0.02mm-1 mm; preferably, the thickness of the bone fiber is 0.05-0.5 mm.

In another preferred embodiment, the spreading area S of the same bone material is 0.9-120 mm²。

In another preferred example, the ratio of the tiled area to the thickness of the same bone material, i.e. the value of S/is 3 to 12000.

In another preferred example, the ratio of the tiling area to the thickness of the same bone material is 9-5000; preferably 9 to 1000; more preferably 9 to 500 or 9 to 300.

In another preferred embodiment, the length range (L range) of the bone material of the same kind is 0.1-200 mm.

In another preferred embodiment, the length of the same bone material is in the range of 0.9-120 mm.

In another preferred example, the average length of the same bone material is 20-100 mm.

In another preferred embodiment, about 50% to 90% of the same bone material has a length L of 30 to 80 mm.

In another preferred embodiment, the width W of the homogeneous bone material is 0.2-10 mm.

In another preferred example, the average length of the same bone material is 20-100 mm.

In another preferred embodiment, about 50% to 90% of the fibrous bone material of the same kind has a length L of 30 to 80 mm.

In another preferred embodiment, the width W of the homogeneous bone material is 0.5-5 mm; more preferably, it is 0.5 to 2 mm.

In another preferred embodiment, the same bone material is dry.

In another preferred embodiment, the homogeneous bone material has a water content of about 1-5 wt%, preferably 1.5-4 wt%.

In another preferred embodiment, the homogeneous bone material is translucent after rehydration, and/or off-white to warm-white.

In a second aspect of the invention, there is provided a method of preparing an allogeneic bone repair material as described in the first aspect, the method comprising the steps of:

(1) providing a pretreated allogeneic bone;

(2) processing the pretreated allogeneic bone into a fibrous shape to obtain a fibrous allogeneic bone;

(3) performing demineralization treatment on the fibrous allogeneic bone obtained in the step (2) to obtain demineralized allogeneic bone; and

(4) cleaning and/or drying the demineralized allogeneic bone obtained in the step (3) to obtain the allogeneic bone repair material.

In another preferred example, in step (4), the washing is performed with PBS, and/or water (preferably pure water).

In another preferred example, in the step (4), the washing is performed 1-20 times with PBS, and then 3-20 times with water.

In another preferred embodiment, in the step (4), the drying is freeze drying.

In another preferred example, in the step (4), the temperature of the freeze drying is-80 ℃ to-20 ℃.

In another preferred example, in the step (4), the freeze-drying time is 1-14 days.

In another preferred example, the pretreated allogeneic bone in the step (1) is obtained from a allogeneic bone raw material through a pretreatment step, and the pretreatment step comprises:

(a) providing an allogeneic bone raw material:

(b) removing soft tissues of the allogeneic bone raw material to obtain the allogeneic bone with the soft tissues removed; and

(c) and carrying out degreasing treatment and virus inactivation treatment on the allogeneic bone with the soft tissue removed to obtain the pretreated allogeneic bone.

In another preferred example, the allogeneic bone material is fresh cadaver bone.

In another preferred example, in the step (c), the degreasing treatment is a degreasing treatment by washing

In another preferred example, in the step (c), the cleaning is ultrasonic cleaning.

In another preferred example, in the step (c), the treatment time of the degreasing treatment is 1 to 7 days.

In another preferred embodiment, the degreasing treatment and the virus inactivation treatment may be performed simultaneously or sequentially.

In another preferred example, the virus inactivation treatment is performed after the degreasing treatment, or the degreasing treatment is performed after the virus inactivation treatment.

In another preferred example, in the step (3), the demineralization treatment is performed by using dilute hydrochloric acid.

In another preferred embodiment, the dilute hydrochloric acid is 0.1M to 1M aqueous hydrochloric acid.

In another preferred example, in the step (3), the treatment time of the demineralization treatment is 0.1-96 hours; preferably, it is 0.1 to 24 hours.

In a third aspect of the invention, there is provided a method of using the homogeneous bone repair material as described in the first aspect, comprising the steps of: mixing the bone repair material and a rehydration liquid to rehydrate, thereby obtaining a rehydrated bone repair material.

In another preferred example, the rehydration liquid is physiological saline.

In another preferred example, the dosage ratio (g: ml) of the bone repair material to the normal saline is 1: (1-10); preferably, the ratio is 1 (3-7).

In another preferred example, the method further comprises: mixing the rehydrated bone repair material with an additional therapeutic agent to form a formulation for bone repair, wherein the additional therapeutic agent is selected from the group consisting of: BMP, cytokines, osteoblasts, stem cells, antibiotics, bone cement, or combinations thereof.

In another preferred example, the method further comprises: mixing the rehydrated bone repair material with autologous blood and/or autologous bone fragments.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In a fourth aspect of the invention, there is provided a method of promoting new bone and/or bone marrow formation, the method comprising the steps of:

(i) mixing the bone repair material and a rehydration liquid to rehydrate to obtain a rehydrated bone repair material; and

(ii) implanting the rehydrated bone repair material into a desired site of a subject.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In a fifth aspect of the invention, there is provided a use of the allogeneic bone repair material for promoting new bone and/or bone marrow formation.

In another preferred embodiment, the allogeneic bone repair material is used for bone defect, bone nonunion and/or bone delayed-healing or nonunion filler repair.

In another preferred embodiment, the allogenic bone repair material is used for spinal fusion, joint fusion and/or orthopedic bone graft repair.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1A shows the effect of the present invention after rehydration of allogeneic bone repair material of varying thickness.

FIG. 1B shows the effect of the allogenic bone repair material of the present invention after rehydration.

FIG. 2 is a graph showing the results of cytotoxicity experiments on homogeneous bone repair materials of different thicknesses and ordinary cancellous bone particles.

FIG. 3 is a diagram of the morphology of co-cultured cells of common cancellous bone particles of different thicknesses of the same bone repair material.

FIG. 4 is a graph showing HE staining results of ectopic osteogenesis of demineralized bone fibers with different thicknesses in nude mice.

Figure 5 shows a nude mouse that has been implanted with a sample of the allograft bone repair material.

FIG. 6 shows a sample of an allograft bone repair material of the invention.

Fig. 7 shows the sample of fig. 6 after rehydration of the allograft material.

FIG. 8 is a scanning electron microscope (X100) of bone fibers of the homogeneous bone repair material of the present invention.

Detailed Description

The inventor has studied extensively and deeply and developed a fibrous allograft bone repair material for the first time. Specifically, the fibrous homogeneous bone material of the homogeneous bone repair material retains the natural microstructure of cortical bone, and each bone fiber contains a large number of hastelloy system structures, so that an excellent microenvironment is created for bone conduction. In addition, the bone repair material of the invention not only retains the structure for promoting bone formation, but also retains some components for promoting bone formation, and some components are exposed on the surface of the bone repair material of the invention or are positioned in a shallow layer, thereby effectively promoting bone formation through bone conduction, bone induction and other modes after being implanted into a bone defect part, promoting the formation of combination with surrounding tissues after finishing creeping substitution, and finally finishing bone repair. The present invention has been completed based on this finding.

Term(s) for

As used herein, the terms "demineralized bone matrix", and "demineralized bone matrix" are used interchangeably to refer to a bone graft material formed from allogeneic bone after a decalcification process, which has reduced immunogenicity.

As used herein, the terms "fibrous" and "tape-like" are used interchangeably to refer to the shape of the allogenic bone repair material according to the first aspect of the invention.

As used herein, the term "tiled area" refers to the area of the allograft bone repair material according to the first aspect of the invention that is taken and tiled.

As used herein, length, width, thickness, and areal extent all refer to the dimensions of the bone repair material as it is not rehydrated.

Allogeneic bone repair material

The invention provides an allogeneic bone repairing material which is fibrous (or called banding) allogeneic bone material.

In a preferred embodiment, as shown in fig. 6, the unhydrated allogeneic bone repair material of the present invention is white with yellow light.

In a preferred embodiment, as shown in fig. 7, after a single strip of homogeneous bone material is rehydrated and spread flat, the flat surface of the homogeneous bone material is substantially rectangular or square (the edges may be flat or uneven, for example, the edges may be burrs or teeth).

Preferably, the length of the flat pavement made of the same bone material is 0.1-200 mm (more preferably, the length is 0.9-120 mm); preferably, the width of the flat pavement is 0.2-10 mm (more preferably, 0.5-5 mm).

The homogeneous bone repair material of the invention can be conveniently subjected to rehydration treatment, has excellent mechanical properties (particularly excellent flexibility) after rehydration, and a plurality of homogeneous bone repair materials can be conveniently formed into various required shapes, thereby being suitable for bone repair parts with various shapes. Typically, a single strip of homogeneous bone material after rehydration is in the form of a ribbon (and in some cases, the length L and width W of the ribbon are substantially the same), and is flexible, freely bendable, translucent, and white. After rehydration and agglomeration, the allogeneic bone repair material is milk white to beige.

The homogeneous bone material has a Hawthorn system structure, wherein the homogeneous bone material comprises a complete or incomplete Hawthorn system structure; more preferably, every 1mm²Cross-sectional homogeneous bone material is usually seen (or exists) in 0.2-10 Haas system structures.

Preparation method of allogeneic bone repair material

The invention provides a preparation method of an allogeneic bone repair material, which comprises the following steps:

removing soft tissue from fresh allograft bone (e.g., cadaveric bone); cleaning (such as 1-7 days) and inactivating viruses to obtain pretreated allogeneic bones;

forming the pretreated allogeneic bone into a fibrous shape (preferably, a length of less than 200mm (e.g., 0.9mm to 120 mm); and/or a thickness of less than 3mm (e.g., 0.01mm to 2mm, preferably, 0.03 to 1 mm; more preferably, 0.05 to 0.7mm)) by machining;

demineralizing fibrous isogenous bone with dilute hydrochloric acid (such as demineralizing for 0.1-96 h);

then washing with PBS (such as washing for 1-20 times);

then cleaning with purified water (for example, cleaning 3 to 20 times)

Freeze drying (at-80 deg.C to-20 deg.C and/or for 1-14 days).

Application of allogeneic bone repair material

The invention provides the use of the allogeneic bone repair material for promoting the formation of new bone and/or bone marrow.

The invention also provides the use of the allogeneic bone repair material for filling repair of bone defects, nonunion and/or delayed or nonunion of bone, and/or for spinal fusion, joint fusion and/or orthopedic bone graft repair.

Application method of allogeneic bone repair material

The use method of the allogeneic bone repair material comprises the following steps: putting the demineralized bone fibers into a sterile vessel, adding a rehydration liquid (such as sterile normal saline) according to the proportion of adding 1g of the product into 5ml of the rehydration liquid for rehydration to form a self-adhesive briquette, and then implanting the briquette into a prepared bone grafting bed.

Preferably, the patient's blood or autologous bone fragments obtained when preparing the bone wound are mixed with the allogenic bone repair material of the invention before implantation to obtain a better result.

The main advantages of the invention include:

(a) the allogenic bone repair material provided by the invention keeps the natural microstructure of cortical bone, each bone fiber contains a plurality of complete and non-complete Hastelloy system structures, and a good microenvironment is created for bone conduction.

(b) The allogenic bone repair material of the invention reserves a large amount of active bone morphogenetic proteins such as BMP, and the bone morphogenetic proteins are positioned on the surface or shallow layer, thereby having excellent bone induction effect and effectively promoting the formation of a large amount of new bones and bone marrow.

(c) The allogeneic bone repair material has very low or almost no immunogenicity, and is not easy to cause immunological rejection.

(d) The homogeneous bone repair material has good rehydration and agglomeration effects, and can conveniently form a shape corresponding to a repaired part.

(e) The allogeneic bone repair material has excellent shaping capacity after rehydration, is suitable for bone repair of different parts, has high rehydration speed (rehydration can be completed within 1-5min generally), has water absorption rate of more than or equal to 300 wt% (far better than granular allogeneic bone repair materials), and has excellent flexibility and mechanical strength after rehydration.

(f) Has good stability, and has a shelf life of at least 3 years at room temperature. Experiments show that compared with a sample which is just prepared, the allogenic bone repair material has no change and adjustment in appearance, water content, calcium content and pH value after being stored for 6 months at room temperature under a sealed condition.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Material

Materials: fresh allogeneic bone