阅读说明：本技术 骨替代材料及其制备方法和应用 (bone substitute material and preparation method and application thereof ) 是由 马克·斯拜克 许和平 迈伦·斯佩克特 范彧 许剑松 于 2019-10-11 设计创作，主要内容包括：本发明涉及一种由β-三磷酸钙和羟基磷灰石双相复合生物陶瓷,含有硅、钙、磷等成分的具有生物活性的生物玻璃和具有三螺旋结构的I型胶原复合而成的骨替代材料,及其制备方法和应用。该新型材料具有免疫抗原性低,生物相容性佳,高性能的成骨作用,以及较长时间的体内存留。将其应用为骨替代材料,在骨缺损、骨创伤特别是脊柱融合术后中使用,能够有效地增加植骨的成骨率,减少患者自身供骨的痛苦与负担,减少二次手术的几率与病人痛苦,为患者谋取更大的福利。(The invention relates to a bone substitute material compounded by beta-calcium triphosphate and hydroxyapatite biphasic compound bioceramic, bioglass with bioactivity containing silicon, calcium, phosphorus and the like and I-type collagen with a triple-helical structure, and a preparation method and application thereof. The novel material has low immunity and antigenicity, good biocompatibility, high-performance osteogenesis effect and long-time in vivo persistence. The material is applied as a bone substitute material, is used after bone defects and bone wounds, particularly spinal fusion, can effectively increase the bone formation rate of bone grafting, reduce the pain and burden of a patient for supplying bone, reduce the probability of secondary operation and the pain of the patient, and bring greater benefit to the patient.)

1. A bone substitute material characterized by: consists of a two-phase composite bioceramic comprising beta-calcium triphosphate and hydroxyapatite, bioactive glass and I-type collagen fibers maintaining a specific triple-helical structure of collagen; wherein the two-phase composite bioceramic and the bioactive glass account for 60-95wt% together, and the type I collagen fiber accounts for 40-5 wt%; the mass ratio of the two-phase composite bioceramic to the bioactive glass is (58-93) to (1.5-3).

2. The bone substitute material of claim 1, wherein: in the two-phase composite bioceramic, the proportion of beta-calcium triphosphate is 70-90wt%, and the proportion of hydroxyapatite is 30-10 wt%; the two-phase composite biological ceramic is granular, the grain size is 0.5-5 mm, the pore diameter is 50-1000 microns, and the porosity is 60-90%.

3. Bone substitute material according to claim 1 or 2, characterized in that: the bioactive glass comprises silicon dioxide (SiO)₂) Sodium oxide (Na)₂O), calcium oxide (CaO) and phosphorus pentoxide (P)₂O₅) The mass ratio of the components is (45 +/-5): (24.5 +/-5): (24.5 +/-5): 6 +/-5); the bioactive glass is spherical particles with the particle size of 150-800 microns; the bioactive glass is preferably 45S 5.

4. The bone replacement material according to any one of claims 1 to 3, wherein: the I type collagen fiber is a medical natural collagen material which takes bovine achilles tendon as a raw material, is extracted to obtain protein with the purity of more than 98 percent and keeps a triple-spiral structure of the I type collagen.

5. A method of preparing a bone replacement material according to any one of claims 1 to 4, the method comprising the steps of:

(1) Treating type I collagen fibers to obtain a concentrated solution of type I collagen fibers;

(2) Sequentially adding the two-phase composite bioceramic and the bioactive glass to the I-type collagen fiber concentrated solution obtained in the step (1) according to a proportion;

(3) Subpackaging the solution obtained in the step (2) into a mould;

(4) Carrying out freeze drying;

(5) And (4) sterilizing and disinfecting the freeze-dried product obtained in the step (4) by using low-temperature ethylene oxide or gamma rays to obtain the bone substitute material.

6. The method of claim 5, wherein: step (1) dissolving type I collagen fibers in 0.0005-0.002 mol/L hydrochloric acid solution to form a suspension with a concentration of 1-4wt%, preferably 0.75-2 wt%; continuously stirring at-2 to 15 ℃ for 30 to 90 minutes at 10,000 revolutions per minute and 20,000 revolutions per minute to homogenize the type I collagen fiber suspension; then concentrating the homogenized solution, namely shaking the solution on a constant speed shaker at the frequency of 25-100 rpm, performing vacuum pumping for 30-60 minutes, and then performing vacuum filtration to obtain a concentrated solution; in this step, the pH value is maintained between 4 and 6 by means of a 0.5 to 2 mol/l hydrochloric acid solution and a 0.5 to 2 mol/l sodium hydroxide solution.

7. The method according to claim 5 or 6, characterized in that: accurately weighing the two-phase composite bioceramic including beta-calcium triphosphate and hydroxyapatite according to a proportion, adding the two-phase composite bioceramic into the I-type collagen fiber concentrated solution within 30-90 seconds, and uniformly stirring; (b) adding 0.5-2 mol/L hydrochloric acid solution into the solution obtained in the step (a) while stirring, and adjusting the pH value of the solution to 4-6; (c) accurately weighing the bioactive glass according to the proportion, gradually adding the bioactive glass into the solution obtained in the step (b) while stirring until the bioactive glass is uniformly mixed; preferably, the step (3) comprises adopting a 316L stainless steel freezing mold, wherein the mold is 350 mm long, 150 mm wide and 3-10 mm thick, the sample grooves are respectively 35x100x5 mm, 28x82x5 mm, 26x62x5 mm, 21x52x5 mm and 20x30x5 mm in size, and the cover plate is 3-10 mm thick and covers a 20-40-mesh high polymer nylon gauze.

8. The method according to any one of claims 5 to 7, wherein: the step (4) comprises (a) a pre-freezing stage, wherein the temperature is set to be-20 to-4 ℃, the time is 30 to 90 minutes, and the state is dynamic or continuous; (b) in the freezing stage, the temperature is set to be-60 to-40 ℃, the time is 120-; (c) a sublimation stage, wherein the temperature is set to be 0 ℃ and the time is 16-30 hours; (d) in the secondary drying and heating stage, the temperature is set to be 20-30 ℃ and the time is 30-90 minutes;

In the freezing process, the temperature of a freezing cabinet of the freeze dryer is kept at minus 80 ℃; the vacuum of the freeze drying apparatus was set at 150 and 300 millitorr.

9. The method according to any one of claims 5 to 8, wherein: after the freeze drying in the step (4), a step of strengthening the crosslinking of the obtained product by a chemical method is further included;

Preferably, the low-temperature ethylene oxide sterilization and disinfection in the step (5) specifically refers to sterilization and disinfection by using ethylene oxide gas, namely 30% -50% of ethylene oxide and 50% -70% of carbon dioxide are used for sterilization in a sterilizer, and the sterilization temperature is 54 ℃ +/-2 ℃; the sterilization humidity is 50-70% RH; the pre-vacuum degree is-20 to-40 Kpa; the ventilation vacuum degree is-40 to-60 Kpa; the number of ventilation times is 8; the sterilization time is 3-4 hours;

preferably, in the gamma ray sterilization process in the step (5), the gamma ray sterilization dose is set to be 20-50 kGy.

10. Use of the bone substitute material according to any one of claims 1 to 4 and the bone substitute material obtained by the method according to any one of claims 5 to 9 for the preparation of a bone fusion, bone filling material.

Technical Field

The invention relates to the technical field of medicine, medical biomaterials, medical tissue engineering and regenerative medicine, in particular to a bone substitute material compounded by beta-calcium triphosphate and hydroxyapatite, bioglass with bioactivity containing silicon, calcium, phosphorus and the like and I-type collagen with a triple helix structure, and a preparation method and application thereof.

background

With the rapid development of the whole medicine in the end of the twentieth century, the progress and development of cell biology, molecular biology, genetic genetics, tissue engineering and regenerative medicine are also continuous, and the biological material with good performance is rapidly discovered and utilized. This has been demonstrated in recent years by the rapid deployment of tissue engineering and regenerative medicine in clinical medicine. Research on bone substitute materials having osteogenic properties in the fields of tissue engineering and regenerative medicine is more widespread and widespread, and significant development has been made internationally in search for the preparation and application of bone substitute materials having better osteogenic effects.

Newer, more effective materials are found among the many bone replacement materials, making them an important research and development topic for use in spinal fusion. The material has the characteristics of more effective osteogenic fusion effect, better biocompatibility, convenient use and the like. In the case of bone diseases causing a wide range of bone defects or in the case of fixation after spinal surgery for maintaining spinal stability, a large number of bone grafting methods and means are indispensable. The bone grafting is most effective and certain in autologous bone grafting, which is also called as gold method, while the bone growth factor (bone morphogenetic protein-2/BMP-2) developed in the eighties of the twentieth century is unprecedented praise due to the use of the type I collagen membrane, and the bone forming/fusion effect of the bone growth factor is comparable to that of the gold bone grafting method.

In the case of a human autologous bone grafting method, if the amount of the required bone is too large or the patient does not have to provide autologous bone, bone source becomes a problem, and the donor site of the patient providing a large amount of autologous bone causes permanent damage and residual pain of the postoperative sequelae. The risk of using bone morphogenetic proteins begins to appear after 20 years of application, and the occurrence probability and the rising trend of the cancers are caused, so that the use standard needs to be reevaluated, and the use amount and the use range are limited.

From the establishment of orthopedics and trauma surgery, osteogenesis is one of the problems to be solved which are very important in the field, and at the same time, research and development of bone substitutes are being synchronously developed, and how to discover or synthesize new osteogenic materials and use has never been stopped in the fields of biomaterial science and biochemistry. It is known from the old that the use of a single simple material, such as calcium sulphate, is used directly in the human body, and the subsequent discovery of the use of a calcium-phosphorus two-phase synthetic material further enhances the use of such materials and achieves good results; the new use of the substances in the form of single molecules to the materials such as hydroxyapatite and the like leads the osteogenic biomaterial to be further leap; the research and use of calcium-phosphorus substances simulating bone components to bioactive bioglass opens up a new field of osteogenesis; the use of single inorganic ceramic minerals in combination with growth factors and stem cells opens up new sections for tissue regulation and regenerative medicine. Wherein, various osteogenic biomaterials are widely accepted and popularized in orthopedics and trauma surgery as bone substitute materials and spine fusion materials with different materials, proportions and manufacturing methods.

Materials with single chemical structures such as calcium sulfate, calcium phosphate and the like are easy to manufacture and obtain, but after the particles of the materials are transplanted in vivo, substances with single components are quickly decomposed in vivo, so that the environment, time and conditions required by bone formation cannot be effectively formed, and the bone formation effect of new bones is poor. The hydroxyapatite polymer material has the structure and the composition of bone-like components, but has very long decomposition time in vivo, even can be as long as several years, so that the long-time degradation rate can influence the bone-forming effect and can also cause other side effects; for materials such as calcium phosphate with improved single chemical substances, such as tricalcium phosphate, etc., the osteogenic effect of the materials is greatly improved compared with that of single-component materials due to the change of the calcium-phosphorus proportion of the materials, but the materials are found to be decomposed faster in vivo in the environment of blood or marrow blood in clinical use, and the materials disappear before effective new bones are formed to lose the high-efficiency osteogenic value, so that the expected effect cannot be achieved.

It is important to design and create a new bone substitute material with high osteogenesis efficiency and better biocompatibility for use in bone substitute materials, particularly for spinal fusion, to provide a better osteogenesis effect. The conditions for osteogenesis required for spinal fusion are more innovative and challenging than typical bone defect filling applications. How to form a sufficiently strong fused bone material in a short time by grafting bone under the dynamic conditions of spinal fusion is a key issue to be solved.

Disclosure of Invention

In order to solve the technical problems, the research on repairing bone defects and spinal fusion is more advanced and prominent by using natural type I collagen as a material carrier and adding substances with good osteogenic performance, such as beta-calcium triphosphate, hydroxyapatite composite biphasic ceramic, active bioglass and the like with special content proportion. The composite material with a specific ratio which is approximately in accordance with the natural ratio of mineral substances to organic substances in human bones is selected, wherein the ratio of the total content of inorganic mineral substances to the type I collagen is in the range of 60-95% to 40-5%, and the ratio of the total content of inorganic mineral substances to the content of organic substances in human bones is closer to the ratio of the content of inorganic substances to the content of organic substances in human bones of 80: 20. The calcium triphosphate and the hydroxyapatite with special proportion are used as a dual-phase ceramic material, and a three-dimensional pore structure with a specific pore size is selected and formed in the material manufacturing process, and the three-dimensional pore structures can enlarge the contact area of the material at the position required by bone grafting in vivo and the surrounding bone tissues, and are favorable for osteoblasts or stem cells to form new bones by utilizing the calcium and phosphorus components of the material. In the process of using the material together with blood or marrow blood, the hydroxyapatite can prolong the absorption and degradation time of tricalcium phosphate and can be kept in vivo for sufficient time to provide inorganic mineral materials required for forming new bones. The use of the solid bioglass microparticles can fully contact osteoblasts by utilizing the existence of the shapes of the microparticles in the pores and the transplanting positions of the tricalcium phosphate/hydroxyapatite double-phase ceramic, enhance the osteogenesis effect by utilizing various chemical components, and simultaneously enable new bone to be firmer. The natural I-type collagen with a triple-helical structure not only has the I-type collagen required by a natural bone matrix, but also can enable the collagen fibers to be uniformly coated with particles carrying double-phase ceramic and active bioglass by compounding the collagen fibers and the inorganic mineral substances, so that the particles can well keep a certain shape, and meanwhile, the built-in material can be kept stable and firm at a bone grafting part, and plays an important role in adsorbing and adhering osteoblasts.

in one aspect the invention relates to a bone substitute material consisting of a biphasic composite bioceramic comprising beta-calcium triphosphate and hydroxyapatite, bioactive glass and type I collagen fibres maintaining the characteristic triple helical structure of collagen. Wherein the two-phase composite bioceramic and the bioactive glass account for 60-95wt% together, and the type I collagen fiber accounts for 40-5 wt%; the mass ratio of the two-phase composite bioceramic to the bioactive glass is (58-93) to (1.5-3).

In a preferred embodiment, the biphasic composite bioceramic has beta-calcium triphosphate in 70-90wt% and hydroxyapatite in 30-10 wt%. The two-phase composite biological ceramic is granular, the grain size is 0.5-5 mm, the pore diameter is 50-1000 microns, and the porosity is 60-90%.

In a preferred embodiment, the bioactive glass comprises silicon dioxide (SiO)₂) Sodium oxide (Na)₂o), calcium oxide (CaO) and phosphorus pentoxide (P)₂O₅) The mass ratio of the components is (45 +/-5): (24.5 +/-5): (24.5 +/-5): 6 +/-5). The bioactive glass is spherical and granular, and has the grain diameter of150-. In a preferred embodiment, the bioactive glass is 45S 5.

The I type collagen fiber is a medical natural collagen material which is extracted from bovine achilles tendon and has the purity of more than 98 percent and maintains the triple-helical structure of the I type collagen; see CN101569765B for a specific preparation method, the relevant contents of the disclosure of which are incorporated herein in their entirety.

In another aspect of the present invention, it relates to a method for preparing a bone replacement material consisting of a biphasic composite bioceramic comprising beta-calcium triphosphate and hydroxyapatite, bioactive glass and type I collagen fibres maintaining the characteristic triple helical structure of collagen, said method comprising the steps of:

(1) treating type I collagen fibers to obtain a concentrated solution of type I collagen fibers;

(2) sequentially adding the two-phase composite bioceramic and the bioactive glass to the I-type collagen fiber concentrated solution obtained in the step (1) according to a proportion;

(3) subpackaging the solution obtained in the step (2) into a mould;

(4) Carrying out freeze drying;

(5) and (4) sterilizing and disinfecting the freeze-dried product obtained in the step (4) by using low-temperature ethylene oxide or gamma rays to obtain the bone substitute material.

In a preferred embodiment, step (1) dissolves type I collagen fibers in a hydrochloric acid solution of 0.0005 to 0.002 mol/l to form a suspension with a concentration of 1 to 4wt%, preferably 0.75 to 2 wt%; continuously stirring at-2 to 15 ℃ for 30 to 90 minutes at 10,000 revolutions per minute and 20,000 revolutions per minute to homogenize the type I collagen fiber suspension; the homogenized solution is then concentrated by shaking on a constant speed shaker at a frequency of 25-100 rpm, vacuum pumping for 30-60 minutes, followed by vacuum filtration to obtain a concentrated solution. In this step, the pH value is maintained between 4 and 6 by means of a 0.5 to 2 mol/l hydrochloric acid solution and a 0.5 to 2 mol/l sodium hydroxide solution.

In a preferred embodiment, the step (2) comprises (a) weighing the biphasic composite bioceramic comprising beta-calcium triphosphate and hydroxyapatite in proportion and adding it to the type I collagen fiber concentrated solution within a period of 30-90 seconds, stirring uniformly; (b) adding 0.5-2 mol/L hydrochloric acid solution into the solution obtained in the step (a) while stirring, and adjusting the pH value of the solution to 4-6; (c) weighing the bioactive glass according to the proportion, gradually adding the bioactive glass into the solution obtained in the step (b) while stirring until the bioactive glass is uniformly mixed.

In a preferred embodiment, the step (3) comprises using a 316L stainless steel freezing mold, wherein the mold has a length of 350 mm, a width of 150 mm and a thickness of 3-10 mm, the sample grooves have dimensions of 35x100x5 mm, 28x82x5 mm, 26x62x5 mm, 21x52x5 mm and 20x30x5 mm, respectively, and the cover plate has a thickness of 3-10 mm and covers a 20-40 mesh polymer nylon gauze. And (3) subpackaging the solution obtained in the step (2) into a sample groove of a freezing mould, wherein no bubble, no bulge, no depression and no obvious gap are required, after filling, placing a gauze on the surface of a filling material, and firmly installing a cover plate on the mould.

In a preferred embodiment, step (4) comprises in particular (a) a pre-freezing phase, set at a temperature ranging from-20 to-4 ℃ for 30 to 90 minutes, in a dynamic or continuous state; (b) in the freezing stage, the temperature is set to be-60 to-40 ℃, the time is 120-; (c) a sublimation stage, wherein the temperature is set to be 0 ℃ and the time is 16-30 hours; (d) and in the secondary drying and heating stage, the temperature is set to be 20-30 ℃ and the time is 30-90 minutes. During the freezing process, the freezer temperature of the freeze dryer was maintained at-80 ℃. In each stage of the step (4), the vacuum degree of the freeze drying equipment is set at 150-300 mTorr.

In a preferred embodiment, after the freeze-drying in step (4), a step of chemically enhancing the crosslinking of the obtained product is further included.

In a preferred embodiment, said low temperature ethylene oxide sterilization of step (5) is described in CN101569765B, the disclosure of which is incorporated herein in its entirety. Specifically, ethylene oxide gas is used for sterilization and disinfection, namely 30% -50% of ethylene oxide and 50% -70% of carbon dioxide are used for sterilization in a sterilizer, and the sterilization temperature is 54 +/-2 ℃; the sterilization humidity is 50-70% RH; the pre-vacuum degree is-20 to-40 Kpa; the ventilation vacuum degree is-40 to-60 Kpa; the number of ventilation times is 8; the sterilization time is 3-4 hours. And (3) sterilizing and disinfecting by using the gamma ray, and setting the sterilization dose of the gamma ray to be 20-50 kGy.

In another aspect the present invention relates to the use of a bone substitute material as described above for the preparation of a bone filling material for bone fusion.

The bone substitute material with good biocompatibility and excellent osteogenesis performance provided by the invention is used for bone filling, is mainly used for spinal fusion, and the prepared product has the thickness of 3-7 mm, the length of 100-25 mm and the width of 20-35 mm.

In the preparation process, I-type collagen fiber turbid liquids with different concentrations are prepared, two-phase composite bioceramic and bioactive glass with different contents are added, and molds with different sizes are selected, so that when the bone substitute material is used, how to select materials with different mineral contents to match with a small amount of autologous cancellous bone to obtain a large amount of effective new cancellous bone can be determined for bone repair and spine fusion. By setting different freeze-drying data parameters, materials with different pore sizes can be obtained. For spine fusion such as non-healing of the lower end of the refractory tibiofibula, giant bone cyst, and spine instability, a bone substitute material containing more minerals is required; for bone grafting or limb facet joint fusion and the like after fracture internal fixation, a small dose of material with mineral content can be selected. The material with the width and the length of 21x52x5 mm is preferred; more preferably a material having a width and a length of 26x62x5 mm; most preferred is a material having a width and length of 28x82x5 mm.

The bone substitute material of the invention is prepared by taking high-purity beta-calcium triphosphate and hydroxyapatite two-phase composite bioceramic with special component proportion, bioactive glass and type I collagen which naturally keeps a triple helical structure as materials, and has the following characteristics: (1) an osteogenic substitute material having 3 osteogenic components; (2) has an osteogenic substitute material closest to the type I collagen and mineral components in human bones; (3) the beta-calcium triphosphate and hydroxyapatite composite mineral with special proportion has a three-dimensional pore structure with special diameter and size; (4) bioactive bioglass containing multiple mineral components; (5) the use of high purity non-soluble native type I collagen fiber material; (5) the sheet material prepared by the non-crosslinking reinforcement or chemical crosslinking reinforcement method can be kept in vivo for a long time, which is beneficial to the formation of new bones; (6) the solid or mud-ash material is easy to fill in bone defect or spine fusion operation, can fully contact the wound surface of a bone grafting part and plays a role in bone formation or spine fusion; (7) the solid bone substitute material formed by the beta-calcium triphosphate, the hydroxyapatite composite ceramic, the bioglass and the type I collagen which naturally keeps a triple-helical structure in a special proportion has high osteogenic performance, good biocompatibility, long-term in-vivo retention and unique plasticity, and is easy to use in an operation.

The use of the I-type collagen material with 98% purity and a triple-helical structure in the material according to the proportion of bone components in a near body can fully play the role of extracellular interstitium, so that minerals in the bone substitute material are uniformly distributed in a collagen carrier, the formation of osteoblasts is accelerated in a favorable biological environment, and the formation of new bones is favorably promoted.

the natural I type collagen material keeping the triple helical structure cooperates with the bone substitute material of a plurality of mineral substances, has low immunity antigenicity, no foreign body reaction, good biocompatibility and long-time in vivo retention, and simultaneously the composite beta-calcium triphosphate and hydroxyapatite material with special diameter and size three-dimensional pores can allow the bone substitute material to have more internal contact surfaces with cells and other substances, so that the bone substitute material is better beneficial to stem cells, osteoblasts contact, migration, adhesion and differentiation and better promotes osteogenesis. Because no crosslinking reinforcement is adopted, the collagen mineral material can be in a plastic mud-ash state when being contacted and mixed with blood and liquid, and is easy to fill and use in special parts such as spines and the like or depressions and the like; the material cross-linked and reinforced by chemical reagents such as formaldehyde has the properties of hardness and non-plasticity, is mainly used for stacking and filling bone defects or placing between vertebral bodies of the spine, and has strong compressive resistance, stability and firmness; the I-type collagen in the material reinforced by chemical cross-linking has stronger function of resisting degradation of collagenase and can exist in vivo for a longer time. The novel bone substitute material for spinal fusion is mainly suitable for spinal fusion in spinal surgery, and is also a preferred material under the conditions that common fractures, bone nonunion and bone defects need bone substitutes.

Drawings

Fig. 1 is an overall appearance view of the bone substitute material of the present invention.

Fig. 2 is a view showing a state in which the bone substitute material of the present invention is mixed with blood.

Fig. 3 is a schematic view of the position of the bone substitute material of the present invention built in an animal experiment.

FIG. 4 is a view showing a phenomenon of new bone formation and fusion by X-Ray after animal experiments.

Fig. 5 is a view showing new osteogenesis by micro-CT after animal experiments.

Fig. 6 is a view of HE tissue slices showing osteogenesis.

FIG. 7 is a section of non-decalcified tissue, stained with trichrome to show views of spinal staged fusion.

Fig. 8 is a schematic diagram of clinical application.

FIG. 9 is a schematic illustration of the effects of spinal fusion.

Fig. 10 is a view of the mold.

Detailed Description

The principles and features of this invention are described in the following examples in conjunction with the accompanying drawings, which are set forth for purposes of illustration only and are not intended to limit the scope of the invention, which is defined solely by the claims appended hereto. Meanwhile, it should be clear that the following described examples are only the best mode for carrying out the invention, and the embodiments described in the summary of the invention can solve the technical problems to be solved by the invention, and achieve the technical effects to be achieved by the invention.