CN114533958B

CN114533958B - Bone tissue defect repairing material with shaping function and preparation method thereof

Info

Publication number: CN114533958B
Application number: CN202210297116.0A
Authority: CN
Inventors: 崔蓉蓉; 张艳勤
Original assignee: Beijing Yh Biomax Biologic Technologies Co ltd
Current assignee: Beijing Yh Biomax Biologic Technologies Co ltd
Priority date: 2022-03-24
Filing date: 2022-03-24
Publication date: 2023-01-20
Anticipated expiration: 2042-03-24
Also published as: CN114533958A

Abstract

The application belongs to the technical field of bone repair materials, and discloses a bone tissue defect repair material with a shaping effect and a preparation method thereof, wherein the repair material comprises collagen fibers and bone powder, the mass ratio of the collagen fibers to the bone powder is 1 to 19 to 1, the collagen fibers are formed by collagen self-assembly, and a peptide chain forming the collagen fibers keeps a triple-helical structure. The repair material is prepared by self-assembling to form collagen fiber, mixing with bone meal, and freeze drying. The repair material can be agglomerated and shaped when absorbing liquid, has certain viscosity, is not easy to disperse, can be completely filled into a bone defect part and keeps the shape, and is particularly suitable for maxillary alveolar bones or alveolar bone multi-arm bone defect parts.

Description

Bone tissue defect repairing material with shaping function and preparation method thereof

Technical Field

The application belongs to the technical field of bone repair materials, and relates to a bone tissue defect repair material with a shaping effect and a preparation method thereof, in particular to repair of a jawbone bone tissue defect.

Background

Bone defects are bone defects caused by bone shortage in pathological or operative processes, bone defects caused in pathological processes, such as comminuted fracture, open fracture and massive bone tissue defects caused by factors such as trauma, inflammation, bone disease and the like, osteonecrosis and abscission caused by inflammation, defects caused by osteonecrosis of a large sheet caused by osteoinfarction or osteoischemic necrosis and the like, which belong to bone defects caused by diseases. The bone defect caused by the operation is caused by artificial factors. Due to the presence of bone defects, nonunion, delayed or no healing of the resulting bone, and localized dysfunction are often caused.

In the oral clinical field, periodontal bone defects are common oral diseases, and mainly include jaw bone defects caused by jaw cyst, tumor, trauma and tooth extraction, alveolar bone absorption caused by periodontal disease, insufficient bone mass in a implanted area and the like. Bone defects tend to be greater than the critical bone defect level in most cases, and the defect area is not sufficient to restore normal bone mass without human intervention. Periodontal tissue regeneration is always a difficult point of periodontal treatment, which includes the reconstruction of cementum, periodontal ligament, alveolar bone and the like, while bone graft regeneration is an important treatment means, which is a common technical means for solving the problem of bone tissue defect.

The bone grafting materials adopted clinically at present mainly comprise autogenous bone, allogeneic bone and synthetic bone substitute materials, and each aggregate has different advantages and disadvantages and needs to be selected by combining specific applicable conditions when in use. Particularly in the oral clinical field, the commonly used bone repair materials are generally granular, the problem that bone powder is scattered due to electrostatic action exists in the use process, and although normal saline or autologous blood can be used for wetting, when multi-arm bone defects exist at a filling site, the conventional bone powder which is wetted is not easy to be stacked and is easy to scatter. Especially, when filling maxillary alveolar bone and multi-arm bone defects, the bone powder is easy to scatter and fall off due to the action of gravity or lack of a supporting surface, so that the operation time is prolonged, and the bone powder is not beneficial to the survival of the bone powder in the defect area and the subsequent covering of an isolating membrane.

Disclosure of Invention

Aiming at the defects that the existing repair material is difficult to maintain the shape, is easy to float and fall off and the like in the repair of periodontal bone defects in the oral cavity, one of the purposes of the application is to provide a bone defect repair material based on collagen and acellular bone, wherein the repair material can be agglomerated and shaped when absorbing liquid (physiological saline or autologous blood), has certain viscosity, is difficult to scatter, can be completely filled into the bone defects and maintain the shape, and is particularly suitable for multi-arm bone defects of alveolar bone.

The other purpose of the application is to provide the preparation method of the bone defect repairing material, which is obtained by independently filling collagen to form fibers, uniformly mixing collagen fiber suspension and granular bone powder with a macroporous structure and a microporous structure, concentrating, and freeze-drying. The repair material prepared by the method has the advantages that the collagen fibers and the mineral substances are uniformly mixed, the repair material has plasticity, can be tightly attached to the defect, and is favorable for osteogenesis.

In order to achieve the technical purpose and effect, the application is specifically realized by the following technical scheme:

as one embodiment of the present application, a bone tissue defect repair material is provided, which comprises collagen fibers and bone powder, wherein the mass ratio of the collagen fibers to the bone powder is 1.

The collagen fiber with the triple-helical structure is mixed with the bone meal to form a fiber and particle composite structure, and compared with the pure bone meal, the shape stability of the material is improved, and the material is not easy to scatter in the using process; meanwhile, the collagen fibers have hydrophilicity, and the material has certain pores due to the adoption of a freeze drying method, and the hydrophilic material and the capillary effect enable the material to have better water absorption capacity, so that liquid can be quickly absorbed and the material can be shaped.

Preferably, the mass ratio of the collagen fibers to the bone meal is 1.

Preferably, the collagen fibers are formed by self-assembly of type I collagen.

The I-type collagen is formed by winding 3 polypeptide chains in a supercoiled manner, is important protein of animal connective tissues, and has certain structural and mechanical properties, such as tensile strength, tensile force, elasticity and the like, so as to achieve the functions of supporting and protecting. And the type I collagen is completed under the action of non-covalent bonds in the self-assembly process of forming a fiber structure, and a cross-linking process of covalent bond connection is not performed, so that the repair material has excellent shaping capacity.

The main components of bone tissue are water, organic matter, inorganic salts, etc., and collagen is the most important structural protein in bone. In vivo, collagen molecules are connected end to end and self-assembled into collagen microfibers in a quarter staggered arrangement, and the microfibers are crosslinked into collagen-based fibers and further woven into fiber bundles to build various tissues in the human body. In the skyHowever, in bone tissues, collagen is the main organic component, which is extracellular matrix secreted by osteoblasts in the osteogenic process, is a scaffold deposited by calcium salt and a promoter for mineralizing bone matrix, and effectively promotes differentiation, migration and adhesion of osteoblasts. The repair material containing the type I collagen component is closer to natural bone, can fully play the role of extracellular matrix of the bone, induces the bone marrow stromal cells to be transformed into osteoblasts, and passes through a receptor alpha ₂ 、β ₁ The signal path mediated by the adhesion kinase increases the adhesion capacity of osteoblasts, promotes the differentiation of the osteoblasts, accelerates the formation of the osteoblasts and promotes the formation of new bones.

The bone powder is selected from acellular bone or synthetic bone, the acellular bone is selected from one of allogeneic bone, acellular bovine bone or acellular porcine bone, and the synthetic bone is selected from one of Hydroxyapatite (HA), tricalcium phosphate (TCP) or multiphase composite ceramic.

Preferably, the bone powder is selected from acellular bone or HA-TCP two-phase composite ceramic.

More preferably, the mass ratio of HA to TCP in the HA-TCP dual-phase composite ceramic is 3.

The acellular bone retains the main components and the structure of extracellular matrix, provides a material basis for repairing bone defect tissues implanted into a body after immunogenicity is removed, and can be used for sites for storing and metabolizing cell attachment, growth factors and the like. Meanwhile, the decellularized bone used for the bone repair material has good biocompatibility, biodegradability and osteoconductivity, has the effect of promoting the proliferation of differentiated cells, and accelerates the formation of osteoblasts together with the type I collagen. The acellular bone also has a three-dimensional structure, and has mechanical strength and mechanical property.

The hydroxyapatite is a main inorganic component of human and animal bones, can be chemically bonded with organism tissues on an interface, has a certain adding degree in vivo, can release ions harmless to the organism, can participate in vivo metabolism, has a stimulation or induction effect on hyperosteogeny, can promote the repair of defective tissues, and shows bioactivity. The tricalcium phosphate has good biodegradability, biocompatibility and biological nontoxicity, and the degraded Ca and P can enter a living body circulatory system to form new bones.

The HA-TCP two-phase composite ceramic comprises two components of hydroxyapatite and beta-tricalcium phosphate, the chemical composition of the HA-TCP two-phase composite ceramic is similar to that of inorganic components of bone tissues, and the HA-TCP two-phase composite ceramic HAs good biocompatibility, bioactivity and safety. In the applied material of the bone tissue engineering scaffold, HA HAs stable chemical property, but is slowly degraded and absorbed, and HAs defects in providing a microenvironment rich in calcium and phosphorus ions for the formation of new bones; the degradation speed of TCP is high, but the degradation has obvious influence on the structure of the bracket material, and the formation of new bone is not facilitated. The HA-TCP two-phase composite ceramic can form a tissue engineering scaffold material with proper degradation/redeposition rate by optimizing the composition ratio and preparation conditions of HA and TCP, thereby promoting the growth of bone tissues and ensuring the structural stability of biological materials.

Meanwhile, on the basis of the characteristics of hydroxyapatite and tricalcium phosphate, the HA-TCP two-phase composite ceramic HAs more stable bone induction characteristics, the chemical composition of the HA-TCP two-phase composite ceramic influences the performance of bone induction by influencing the degradation and redeposition rate of calcium and phosphorus, and the physical structure of the two-phase calcium phosphate ceramic further induces the bone differentiation of stem cells to influence the bone induction of the two-phase calcium phosphate ceramic by influencing the adsorption of bone formation related proteins, blood vessel production, tissue ingrowth, local microenvironment and the like. The HA-TCP two-phase composite ceramic HAs the characteristics of good bone induction and bone conduction by combining with the type I collagen.

The bone meal is granules with large pores and microporous structures, and has high porosity, wherein the bone meal porosity is 50-90%, the pore size distribution is 0.01-1500 mu m, and the particle size is 0.1-3.0 mm.

It is generally believed that the structure of the macropores of several hundred micrometers mainly affects the amount of induced osteogenesis, and that micropores, especially micropores of several micrometers and several hundred nanometers, are the key factors determining the osteoinductive properties of the material. The macroporous structure can maintain the shape and mechanical property of the material and support the ingrowth of cells and blood vessels, simultaneously provides necessary structural foundation for the formation and maintenance of local osteogenesis inducing microenvironment in the material, and the micropores allow body fluid to permeate into the implant so as to enhance the biological activity of the implant. The micropores on the surface of the material can be used as key factors for regulating and controlling the degradation and redeposition of the material and promoting the osteoinductivity of the material. The more micropores, the greater the specific surface area of the material and therefore the more proteins adsorbed on the surface, and the further the specific surface area of the pores may increase with degradation of the material and redeposition of the apatite layer. The bone formation related protein adsorbed along with the process can act with stem cells and induce the osteogenic differentiation of the stem cells, thereby promoting the generation of new bone. Meanwhile, the rough surface on the pore structure can have direct effect on the stem cells, and the directional differentiation of the stem cells is induced by influencing the adhesion form of the stem cells.

The particle size of the bone meal is preferably 0.25 to 2.0mm, more preferably 0.3 to 1.0mm. The bone meal has proper particle size and alveolar bone defect size caused by tooth extraction or trauma, and is more favorable for uniform mixing with collagen fibers.

The length of the collagen fiber formed in the repair material is 0.1-5 mm.

The length of the collagen fiber and the particle size of the bone meal particles are kept slightly larger or slightly smaller, so that a uniformly dispersed solution is easier to form when the collagen fiber and the bone meal are mixed, the bone meal and the collagen fiber in the obtained repair material are mutually staggered and uniformly distributed, and the collagen fiber and the bone meal can exert respective characteristics after being tightly attached in the bone defect repair process to promote osteogenesis.

The application bone tissue defect repair material forms collagen fiber turbid liquid through collagen self-assembly, adds bone meal and then stirs, concentrates and stereotypes, and collagen fiber and bone meal misce bene in the repair material who obtains.

As a second embodiment of the present application, there is provided a method for preparing the bone tissue defect repair material, including: a semi-transparent collagen suspension formed by uniformly dispersing collagen in an acid or alkali solution, wherein the fibrous collagen keeps a complete triple-helical structure;

adding bone meal into the semitransparent collagen suspension, stirring, adjusting the pH value to an isoelectric point, and self-assembling to obtain a mixed suspension of white short fibers and the bone meal;

a step of concentrating the mixed suspension by centrifugation or filtration to provide a concentrate; and

and (3) freezing and drying the concentrate after shaping.

The collagen with the spiral structure is added into an acid solution and is subjected to self-assembly in a buffer solution, the process goes through two stages of nucleation and fiber growth, and finally the collagen fiber with the D-band characteristic structure is formed, and the collagen fiber has good cell adhesion, biocompatibility and biodegradability, and particularly becomes a material with unique advantages and potentials in the fields of biomedicine and biosensors through self-assembly behaviors.

The freeze drying is to freeze and freeze the water in the obtained shaped product, and after the pressure is reduced, the frozen ice crystals are directly sublimated, and the ice crystals are changed from solid state to gaseous state to remove water. Compared with the common spray drying technology, the collagen is not denatured without the process of high-temperature water removal, and the activity of the collagen as intercellular substance is kept. Meanwhile, moisture is removed through sublimation, the porous characteristic of the repair material is kept, and the water absorption and shaping performance of the repair material is improved.

Further, the acid solution is selected from one or more of hydrochloric acid, acetic acid, lactic acid solution or citric acid; the alkali solution is selected from NaOH and NaHCO ₃ 、Na ₂ CO ₃ Or KOH solution.

Further, the mass content of collagen in the semitransparent collagen suspension is 0.5-3.5%. The collagen fiber suspension formed under the concentration can be fully and uniformly mixed with the bone meal, and the slurry with too low or too high concentration is not beneficial to forming a homogeneous material with the bone meal.

Further, the acid solution or the alkali solution is adopted for adjusting the pH value, wherein the concentration of the acid solution or the alkali solution is 0.1-1.0 mol/L. In the scheme, the acid or alkali solution is used for adjusting the pH value of the semitransparent collagen suspension to be near the isoelectric point, so that the self-assembly process is accelerated.

Furthermore, the stirring speed is 1-12krpm in the self-assembly process, the pH is adjusted to 4-7.8, and the temperature is 10-25 ℃.

In the scheme, the self-assembly speed and the self-assembly density of the collagen can be controlled by controlling the concentration, the stirring speed, the pH value and the temperature of the collagen, and the length of the collagen fiber is kept between 0.1 and 5mm. Meanwhile, the parameters in the preparation conditions are changed, and the distribution occupancy of the fiber length in the range of 0.1-5 mm can be adjusted, thereby meeting the condition of slightly larger or smaller particle size of the bone meal particles.

Further, the mixture of fiber and mineral matter is concentrated by centrifugation after filtration to reduce the water content of the initial collagen slurry to 96.5-99.5% to 20-70% of the collagen mineral matter.

Further, the freeze drying specifically comprises: cooling to 0-4 deg.c and maintaining for 30-60 min; continuously cooling to-20 to-40 ℃, the cooling rate is 0.5 to 1.5 ℃/min, and the temperature is kept for 60 to 180min; heating to 0 ℃, keeping the pressure not higher than 0.3mbar, and drying in vacuum for 10-30 h; heating to 20-30 deg.c and maintaining for 30-180 min. Before the freeze drying process is started, the mixture is subpackaged according to the amount of 0.5-2.0 g, and the mass of the freeze-dried product is 0.2-0.5 g.

The freeze drying process is divided into four stages, the first stage is to pre-cool the material at the temperature close to 0 ℃, and the structural defects caused by overlarge temperature difference and overlarge crystal structure difference of the material in the rapid cooling process are avoided; in the second stage, the temperature is slowly reduced to-40 ℃ to complete the icing process of the collagen fiber suspension, so that the complete icing in the collagen fiber is ensured, and a precondition is provided for completely removing the water in the subsequent sublimation process; in the third stage, the ice crystals are sublimated by adjusting the pressure at 0 ℃ to complete the water removal process; and in the fourth stage, under the condition that the denaturation temperature is not exceeded, the activity of the collagen fibers is ensured, and meanwhile, residual water is removed, so that the collagen fibers are completely dried.

The beneficial effect of this application does:

1) This application utilizes collagen and bone meal to pass through the self-assembling mode and mixes, control reaction condition makes collagen carry out the self-assembling and forms the fibre and obtain collagen and bone meal combined material, the fibrous structure of because fibrous collagen has produced stronger bonding reunion effect to the bone meal, simultaneously because use freeze drying shaping, the hydrophilicity of fibrous collagen and the porous characteristic of aggregate have promoted repair materials's water absorption and moulding ability, when upper jaw alveolar bone and the damaged department of multi-arm bone have effectively been solved, because the effect of gravity or lack the holding surface and easily scatter the problem that drops.

2) The repairing material contains mineral bone powder and fibrous collagen, has the components closer to natural bone, can fully play the role of extracellular matrix of the repairing material, induces bone marrow stromal cells to be transformed into osteoblasts, promotes the differentiation of the osteoblasts, accelerates the formation of the osteoblasts, promotes the formation of new bone, and has good biocompatibility, biodegradability, osteoconductivity and osteoinductivity.

3) The fibrous collagen and the bone powder with the three-dimensional structure in the repair material also have the function of promoting hemostasis, have good water absorption, can absorb liquid with the weight not less than 1 time of the self weight, are favorable for the wound to form a blood clot and promote the wound healing.

4) Collagen fibers are formed by self-assembly of collagen, the structure of the collagen fibers is closer to that of collagen in vivo, and the good cell adhesion, biocompatibility and compatibility of the collagen are kept. It mixes with the bone meal when forming collagen fiber through self-assembly, compares and makes collagen fiber earlier and then mix with the bone meal, and among the repair material that this application technical scheme made, fibre and mineral substance dispersion are more even. Meanwhile, the length and thickness of the fiber can be controlled by controlling the self-assembly condition so as to match bone meal with different particle sizes to prepare products.

5) The four-section freeze drying process is adopted, high-temperature treatment is not carried out, the activity of collagen serving as intercellular substance is kept, the fibrous tissue structure of the collagen is not damaged, the porous characteristic of the collagen is kept, and the water absorption and the shaping of the repair material are improved.

Drawings

FIG. 1 shows the plastic properties of the prosthetic material of the present application immersed in normal saline; wherein A is the repair material of example 1, and B is the repair material of comparative example 1.

Detailed Description

The technical solutions of the present application will be described clearly and completely with reference to specific embodiments of the present application, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

In one embodiment of the present application, there is provided a bone tissue defect repair material comprising fibrous collagen and bone powder having a helical structure, wherein the mass ratio of fibrous collagen and bone powder is 1. Preferably, the mass ratio of fibrous collagen to bone powder is 3. The repair material with the agglomeration effect is formed by self-assembling the fibrous collagen and the bone meal, and the repair material has macropores, micropores and a fibrous structure, so that the defect that the repair material is easy to disperse and fall off due to the action of gravity or lack of a supporting surface when the repair material is filled in maxillary alveolar bone and multi-arm bone defects is overcome.

Specifically, the fibrous collagen having a spiral structure may be type I collagen, type II collagen, type III collagen, type VII collagen, type X collagen, type XI collagen, type XII collagen, type XIV collagen, type XVII collagen, or the like. In the present embodiment, type I collagen is preferred, and is the collagen that is most abundant in the human body, and can act on extracellular matrix molecules, cell surface molecules, growth factors, differentiation factors, and the like, and has an important role in physiological processes of the body.

In this embodiment, the type I collagen may be obtained by commercially available products, or may be extracted from pigskin, cowskin, bovine tendon, rat tail tendon, fish skin, and bovine achilles tendon, and the extraction process is performed by a technique commonly used by those skilled in the art, for example: acid method, enzyme method, alkali method and salt method, preferably collagen extracted from bovine Achilles tendon.

Specifically, the bone powder is selected from decellularized bone or synthetic bone, the decellularized bone can be allogenic bone, decellularized bovine bone or decellularized porcine bone, and the synthetic bone can be hydroxyapatite, tricalcium phosphate or multiphase composite ceramic. The bone powder has a granular structure with large pores and micro pores, and has a porosity of 50-90%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, a pore size distribution of 0.01-1500 μm, and a particle size of 0.1-3.0 mm. The decellularized bone can be decellularized by using a common decellularization solution consisting of pancreatin, an activating agent and a degreasing agent. The hydroxyapatite can be prepared by a sol-gel method, a hydrothermal method, a precipitation method or a dry solid-phase reaction and a microemulsion method. The multiphase composite ceramic may be a hydroxyapatite- β -tricalcium phosphate-alumina three-phase composite bioceramic or a hydroxyapatite- β -tricalcium phosphate two-phase composite ceramic, and in this embodiment, is preferably a Hydroxyapatite (HA) - β -tricalcium phosphate (β -TCP) two-phase composite ceramic, wherein the mass ratio of HA to TCP is from 3.

In another embodiment of the present application, there is provided a method for preparing the above bone tissue defect repair material, comprising:

preparing collagen slurry: adding the collagen with the triple-helical structure into an acid solution with the pH value of 1.0-3.0, starting a dispersion machine after 5-60 minutes, homogenizing for 5-90 minutes under the condition of 5-20 krpm, wherein the temperature is not higher than 25 ℃, and preparing into translucent jelly-like collagen slurry with the collagen mass concentration of 0.5-3.0%, preferably 0.7-2%. With increasing concentration, the rate of self-assembly can be increased, resulting in a denser fibrous structure.

Preparation of a white collagen fiber suspension: adding bone powder into translucent collagen slurry, regulating the pH value of the translucent collagen suspension to be near the isoelectric point by 0.1-1.0 mol/L alkali solution, continuously stirring for 5-30 min at the rotation speed of 1-12 krpm, at the temperature of 10-25 ℃, performing self-assembly on collagen until the collagen is in a white short fiber shape, and controlling the fiber length to be 0.1-5 mm to obtain the suspension of the white collagen fiber and the bone powder.

And (3) freeze drying: concentrating by filtering or centrifuging to remove liquid from the white collagen fiber suspension, reducing water content to 20-65%, filling the collagen-mineral bone powder mixture into a mold, and freeze drying. The method comprises the following specific steps: cooling to 0-4 deg.c and maintaining for 30-60 min; continuously cooling to-20 to-40 ℃, the cooling rate is 0.5 to 1.5 ℃/min, and the temperature is kept for 60 to 180min; heating to 0 ℃, keeping the pressure not higher than 0.3mbar, and drying in vacuum for 10-30 h; heating to 20-30 deg.c and maintaining for 30-180 min.

And (3) sterilization: the sterilization is carried out by using ethylene oxide, gamma ray and low-temperature plasma, and preferably gamma ray is adopted for sterilization.

The following will explain the repair material of the present application in detail with reference to specific preparation examples.

Example 1

The embodiment provides a preparation method of a bone tissue defect repair material, which comprises the following steps:

1) Adding the I-type collagen extracted from the bovine Achilles tendon into a hydrochloric acid solution with the pH value of 1.5 +/-0.3, soaking for 15-60 min, starting a dispersion machine, and homogenizing at 15-18 krpm for 35min to prepare a semitransparent I-type collagen suspension with the mass concentration of 1.5%.

2) Adding the HA-TCP two-phase ceramic into the suspension according to the mass ratio of the type I collagen to the HA-TCP two-phase ceramic of 1. Wherein, the mass ratio of HA to TCP is 1, the particle size of HA-TCP dual-phase ceramic is 0.2-0.8 mm, the porosity is 70-80%, and the pore size distribution is 0.01-1500 μm.

3) Then, a centrifuge is used for centrifugation at 1000-3000rpm for 1-10 min at the temperature of 20 ℃, and supernatant liquid is removed.

4) And (3) putting the lower-layer precipitate into a mold for freeze drying, firstly cooling to 2 ℃, keeping for 30min, continuously cooling to-40 ℃, cooling at the speed of 0.5-1.5 ℃/min, keeping the temperature for 60-120 min, heating to 0 ℃, keeping the pressure not higher than 0.3mbar, drying in vacuum for 10-20 h, heating to 20-30 ℃, and keeping for 30-90 min.

5) Sterilizing with gamma ray.

Example 2

1) Adding the I-type collagen extracted from the cowhide into a hydrochloric acid solution with the pH value of 1.5 +/-0.3, soaking for 15-60 min, starting a dispersion machine, and homogenizing for 10min at 15-18 krpm to prepare a semitransparent I-type collagen suspension with the mass concentration of 1%.

2) Adding the HA-TCP double-phase ceramic into the suspension according to the mass ratio of the type I collagen to the HA-TCP double-phase ceramic of 1 to 3, stirring at the rotating speed of 1-12 krpm, adding 0.5mol/L KOH solution into the semitransparent type I collagen suspension, and adjusting the pH to 7.5 to obtain a white short fiber and mineral substance mixture. Wherein, the mass ratio of HA to TCP is 1, the particle size of the HA-TCP dual-phase ceramic is 2.0-2.5 mm, the porosity is 70-80%, and the pore size distribution is 0.01-1500 μm.

3) The supernatant was then removed by centrifugation at 500rpm for 20min using a centrifuge at a temperature of 20 ℃.

5) Sterilizing with gamma ray.

Example 3

1) Adding the I-type collagen extracted from the cowhide into a hydrochloric acid solution with the pH value of 1.5 +/-0.3, soaking for 15-60 min, starting a dispersion machine, and homogenizing for 20min at 15-18 krpm to prepare a semitransparent I-type collagen suspension with the mass concentration of 1.3%.

2) Adding the HA-TCP double-phase ceramic into the suspension according to the mass ratio of the type I collagen to the HA-TCP double-phase ceramic of 1 to 9, stirring at the rotating speed of 1 to 12krpm, and using 0.5mol/L of Na ₂ CO ₃ Adding translucent type I glue into the solutionAdjusting the pH value of the original suspension to 7.8 to obtain a white short fiber and mineral mixture. Wherein, the mass ratio of HA to TCP is 1.

3) The supernatant was then removed by centrifugation at 500rpm for 15min using a centrifuge at a temperature of 20 ℃.

4) And filling the lower-layer precipitate into a mold for freeze drying, firstly cooling to 2 ℃, keeping for 30min, continuously cooling to-40 ℃, cooling at the speed of 0.5-1.5 ℃/min, keeping the temperature for 60-120 min, heating to 0 ℃, keeping the pressure not higher than 0.3mbar, vacuum drying for 10-20 h, heating to 20-30 ℃, and keeping for 30-90 min.

5) Sterilizing with gamma ray.

Example 4

1) Adding the I-type collagen extracted from the bovine Achilles tendon into a hydrochloric acid solution with the pH value of 1.5 +/-0.3, soaking for 15-60 min, starting a dispersion machine, homogenizing for 40min at 13-15 krpm, and preparing a semitransparent I-type collagen suspension with the mass concentration of 1.6%.

2) Adding the HA-TCP biphasic ceramic into the collagen suspension according to the mass ratio of the type I collagen to the HA-TCP biphasic ceramic of 1 ₃ Adding the solution into a semitransparent I type collagen suspension to adjust the pH value to 4.8, and obtaining a mixture of white short fibers and substances. Wherein, the mass ratio of HA to TCP is 1.

3) The supernatant was then removed by centrifugation at 1krpm for 10min using a centrifuge at a temperature of 20 ℃.

5) Sterilizing with gamma ray.

Example 5

1) Adding the I-type collagen extracted from the bovine achilles tendon into a hydrochloric acid solution with the pH value of 1.5 +/-0.3, soaking for 15-60 min, starting a dispersion machine, and homogenizing for 60min at 13-15 krpm to prepare a semi-transparent I-type collagen suspension with the mass concentration of 2%.

2) Adding the HA-TCP double-phase ceramic into the collagen suspension according to the mass ratio of the type I collagen to the HA-TCP double-phase ceramic of 1 to 19, stirring at the rotating speed of 1-12 krpm, adding 0.5mol/L NaOH solution into the semitransparent type I collagen suspension to adjust the pH value to 5.0, and obtaining a white short fiber and mineral mixture. Wherein, the mass ratio of HA to TCP is 3.

3) The supernatant was then removed by centrifugation at 1.5krpm for 10min using a centrifuge at a temperature of 20 ℃.

5) Sterilizing with gamma ray.

Example 6

1) Adding the I-type collagen into acetic acid with the pH value of 2.0-3.0, starting a dispersion machine, homogenizing at 13-17 krpm for 30min, and preparing translucent I-type collagen suspension with the mass concentration of 0.7%.

2) Adding the HA-TCP double-phase ceramic into the white short fiber suspension according to the mass ratio of the type I collagen to the HA-TCP double-phase ceramic of 1. Wherein, the mass ratio of HA to TCP is 3, the grain diameter of the HA-TCP two-phase ceramic is 0.1-0.5mm, the porosity is 50%, and the pore size distribution is 0.01-1500 μm.

3) The supernatant was removed by centrifugation at 5krpm for 1-3 min at 10 ℃ using a centrifuge.

4) And filling the lower-layer precipitate into a mold for freeze drying, firstly cooling to 0 ℃, keeping the temperature for 30-60 min, continuously cooling to-40 ℃, keeping the cooling rate at 0.5-1.5 ℃/min, keeping the temperature for 60-180 min, heating to 0 ℃, keeping the pressure not higher than 0mbar, drying in vacuum for 10-30 h, heating to 20-30 ℃, and keeping the temperature for 30-180 min.

5) Sterilizing with gamma ray.

Example 7

1) Adding the I-type collagen into hydrochloric acid with the pH value of 2.0-3.0, starting a dispersion machine, homogenizing at 18-22 krpm for 90min, and preparing semi-transparent I-type collagen suspension with the mass concentration of 3%.

2) Adding the HA-TCP double-phase ceramic into the semitransparent type I collagen suspension according to the mass ratio of the type I collagen to the HA-TCP double-phase ceramic of 1 ₃ Adding the solution into a semitransparent I type collagen suspension to adjust the pH to 4.4, and obtaining a white short fiber and mineral mixture. Wherein, the mass ratio of HA to TCP is 7, the grain diameter of the HA-TCP two-phase ceramic is 1.5-3.0mm, the porosity is 90%, and the pore size distribution is 0.01-1500 μm.

3) The supernatant was removed by centrifugation at 16krpm for 3-6 min at 25 ℃ using a centrifuge.

4) And (3) filling the lower-layer precipitate into a mold for freeze drying, firstly cooling to 0 ℃, keeping the temperature for 30-60 min, continuously cooling to-40 ℃, keeping the cooling rate at 0.5-1.5 ℃/min, keeping the temperature for 60-180 min, heating to 0 ℃, keeping the pressure not higher than 0.3mbar, drying in vacuum for 15-20 h, heating to 20-30 ℃, and keeping the temperature for 120min.

5) Sterilizing with gamma ray.

Example 8

1) Adding the I-type collagen into citric acid solution with the pH value of 2.0-3.0, starting a dispersion machine, and homogenizing at 10-20 krpm for 120min to prepare translucent I-type collagen suspension with the mass concentration of 1%.

2) Adding the HA-TCP double-phase ceramic into the collagen suspension according to the mass ratio of the type I collagen to the HA-TCP double-phase ceramic of 3. Wherein, the mass ratio of HA to TCP is 7, the grain diameter of the HA-TCP two-phase ceramic is 0.2-0.6 mm, the porosity is 50-60%, and the pore size distribution is 0.01-1500 μm.

3) Centrifuging at 1000-3000 rpm for 3-10 min at 20 deg.c and removing the supernatant.

4) And filling the lower-layer precipitate into a mold for freeze drying, firstly cooling to 0 ℃, keeping the temperature for 30-60 min, continuously cooling to-40 ℃, keeping the cooling rate at 0.5-1.5 ℃/min, keeping the temperature for 60-180 min, heating to 0 ℃, keeping the pressure not higher than 0.3mbar, drying in vacuum for 10-30 h, heating to 20-30 ℃, and keeping the temperature for 30-180 min.

5) Sterilizing with gamma ray.

Example 9

1) Adding the I-type collagen into a lactic acid solution with the pH value of 2.0-3.0, starting a dispersion machine, homogenizing at 10-20 krpm for 180min, and preparing translucent I-type collagen suspension with the mass concentration of 2.5%.

2) Adding the HA-TCP double-phase ceramic into the collagen suspension according to the mass ratio of the type I collagen to the HA-TCP double-phase ceramic of 3. Wherein, the mass ratio of HA to TCP is 7.

3) Centrifuging at a temperature of 20 ℃ and a speed of 1000-3000 rpm for 3-10 min by using a centrifuge, and removing supernatant.

4) And filling the lower-layer precipitate into a mold for freeze drying, firstly cooling to 0 ℃, keeping for 30-60 min, continuously cooling to-40 ℃, cooling at the rate of 0.5-1.5 ℃/min, keeping the temperature for 60-180 min, heating to 0 ℃, keeping the pressure not higher than 0.3mbar, vacuum drying for 10-30 h, heating to 20-30 ℃, and keeping for 30-180 min.

5) Sterilizing with gamma ray.

Comparative example 1

The preparation method of the bone tissue defect repair material is the same as the method in example 1, and is characterized in that the semitransparent type I collagen suspension and the HA-TCP two-phase ceramic suspension are directly centrifugally concentrated without a self-assembly process regulated by alkali liquor.

Comparative example 2

A preparation method of the bone tissue defect repairing material is the same as that of the embodiment 1, and is characterized in that after white short fiber suspension is prepared near an isoelectric point by a dispersion machine, the white short fiber suspension is concentrated to high-concentration fiber suspension, then the concentrated collagen fiber suspension is physically mixed with HA-TCP two-phase ceramic or acellular bone powder, and then the mixture is freeze-dried.

Comparative example 3

The preparation method of the bone tissue defect repair material is the same as the method of the embodiment 1, and is characterized in that gelatin is adopted to replace type I collagen, the gelatin is directly mixed with HA-TCP double-phase ceramic, and the mass ratio of the gelatin to the HA-TCP double-phase ceramic is 4.

The properties of the repair material of the present application will be further described with reference to specific effect examples.

Example 10 fiber Length

The white short fiber suspensions formed by self-assembly in examples 1 to 9 were used as an experimental group, and the translucent type I collagen suspension in comparative example 1 was used as a control group, and the fiber length distributions of the experimental group and the control group were measured on a slide glass by observation and measurement under a 4-fold optical microscope.

Table 1 fiber length distribution (%)

As can be seen from Table 1, the fiber length of 95% or more of the white short fiber suspensions of examples 1 to 9 was maintained between 0.1 mm and 5mm, and the fiber length of 65% to 77% of the translucent type I collagen suspension of comparative example 1, which had not been self-assembled, was greater than 5mm. The collagen is added into the acidic liquid to expand, the dispersion machine breaks the macroscopic structure of the collagen to homogenize the collagen into semitransparent gel, the self-assembly rate of the collagen, the fiber concentration and the fiber length and thickness can be controlled by controlling the concentration, the stirring speed, the pH, the temperature and the time of the collagen slurry, the collagen is self-assembled under the action of the pH and the stress, and the collagen is gathered into white short fibers from the semitransparent state. In bone repair materials, one of the roles of the fibers is to encapsulate and agglomerate the mineral particles, preventing them from escaping after delivery or implantation into the defect. The appropriate fiber length can wrap the bone meal particles, the bone meal can be kept agglomerated after being soaked in liquid, the bone meal is kept agglomerated, and the bone meal is prevented from scattering and falling in the using process.

Example 11 repair Material Density Properties

1) Effect of dosage of fibrous collagen and bone meal on Density

Placing the type I collagen in a hydrochloric acid solution to form a semitransparent type I collagen suspension by adopting the method of example 1, adding HA-TCP double-phase ceramic with the particle size of 1-3 mm, mixing, adjusting the pH value by using a NaOH solution, carrying out self-assembly, carrying out centrifugal concentration, subpackaging, and carrying out freeze drying to prepare the repair material. The freeze-dried samples were collectively taken out from the freeze-drying mold, the length, width and height thereof were measured (the cylindrical samples were measured for the bottom diameter and height), the volume was calculated, the weight of the samples was measured using an analytical balance, and the density was calculated.

TABLE 2 Density of repair materials

As shown in table 2, the higher the bone powder content in the bone repair material, the higher the density of the repair material, because the collagen absorbing liquid expands, and the more collagen contained, the larger the volume occupied by the collagen, so that the density of the bone powder after freeze-drying varies with the same process parameters and different specific gravities of the bone powder.

2) Influence of fiber length and bone meal size on Density

The densities of the repair materials formed by self-assembly of HA-TCP two-phase composite ceramics with different particle sizes and collagen fibers with different lengths are measured under the condition that the mass ratio of the collagen to the HA-TCP two-phase composite ceramics is 1.

TABLE 3 Density of repair materials of different fiber lengths and bone meal sizes

As can be seen from table 3, when the size of the bone meal particle size is not much different from the size of the fiber length, the obtained bone meal density data is better because the length of the collagen fiber and the size of the bone meal particle size are kept slightly larger or smaller, when the bone meal is mixed with the bone meal, a uniformly dispersed solution is more easily formed, and the bone meal and the collagen fiber in the obtained repair material are mutually staggered and uniformly distributed.

3) The bone density of example 1 and the ratios 1, 2 and 3 were measured by the same method.

TABLE 4 Density of repair materials

As can be seen from table 4, the density of the material prepared in example 1 was higher than that of comparative example 2, comparative example 1 and comparative example 3 in this order. The reason is that the collagen is in a swelling colloid in the acid solution without the self-assembly process in the comparative example 1, the amount of the removed liquid after the mixed bone powder is centrifuged is far lower than that in the example 1, namely, when subpackaging is carried out before freeze drying, the moisture content in the mixture is higher, the bone powder content is relatively lower, the density after freeze drying is lower than that in the example 1, and the weight fluctuation of the quality inspection of each freeze-dried sample is larger. Comparative example 2 collagen type I was used as a raw material, but collagen fibers were homogenized in a solution near the isoelectric point directly without self-assembly, and collagen was always in a fibrous state, and the assembly process from fibril to macroscopic visible fiber was not performed, and when the collagen fibers were mixed with bone powder, the fibers were easily aggregated, and the bone repair material after freeze-drying was packed separately, and the specific gravities of the bone powder and collagen were unstable, showing that the density of the bone repair material produced by this method had large fluctuations. Comparative example 3 is gelatin, which is a degradation product of collagen, whose triple helix structure has been destroyed, biological activity is reduced, and the biological function of collagen to promote blood coagulation is lost.

EXAMPLE 12 moldability of prosthetic materials

Physiological saline was prepared at a concentration of 0.9%, and the prosthetic materials of example 1 and comparative example 1 were immersed in physiological saline and observed after being maintained for 10 seconds, respectively.

As a result, as shown in FIG. 1A, the prosthetic material obtained in example 1 was kneaded and deformed after being soaked in a physiological saline solution, and lifted up in the air with forceps to maintain its intact appearance without scattering. The material prepared in comparative example 1, as shown in fig. 1B, was soaked in physiological saline and lifted with forceps and then fell out of shape. The collagen I in the product of the embodiment 1 has a good fiber structure after self-assembly, can generate an agglomeration effect after absorbing liquid physiological saline, and is simultaneously uniformly mixed with bone meal in the self-assembly process, so that the fiber structure is fully contacted with the bone meal to have a strong adsorption and agglomeration effect on the bone meal, and the filler is kept to be still capable of keeping a basic form after being wetted.

Example 13 liquid absorption Properties

Experimental materials:

the repairing material prepared by the method of the embodiment 1 is used as an experimental group;

the repair materials prepared by the methods of comparative example 1, comparative example 2 and comparative example 3 were used as control groups.

The experimental method comprises the following steps:

respectively taking equal-volume samples (1 cm multiplied by 0.5 cm) of the experimental group and the control group, placing the samples in a drier, balancing the samples at room temperature for 48 hours, taking out the samples, weighing the samples, and recording the mass as m ₁ And the decimal point is accurate to four digits. Putting the sample into 0.9% normal saline for 2 minutes, taking out, placing on filter paper for 30S to remove surface moisture, and weighing the sample mass m ₂ And the decimal point is accurate to four digits. The experimental and control groups were set up in triplicate per sample.

The absolute water absorption of the sample was calculated as follows:

m _a ＝m ₂ -m ₁

wherein, the first and the second end of the pipe are connected with each other,

m _a the absolute water absorption of the sample;

m ₁ the mass of the sample before soaking;

m ₂ the quality of the test piece after immersion in water.

The percent water absorption of the sample was calculated as follows:

wherein the content of the first and second substances,

m _p is the percentage of water absorbed relative to the initial mass of the sample.

The results are shown in Table 6.

TABLE 6 liquid adsorption Performance results

As can be seen from Table 6, the average water absorption of the product of example 1 was smaller than that of comparative example 2, comparative example 1 and comparative example 3 in this order. The product of the embodiment 1 has a good fiber structure after the I-type collagen is self-assembled, the short fiber structure and the bone meal are mutually staggered and uniformly distributed, a strong adsorption and coagulation effect is generated, and the fibers expand after absorbing water to prevent the fluidity of water, so that the molding capacity of the material is ensured, and the viscosity is low.

Example 14 biocompatibility

The materials of examples 1-9 and comparative examples 1-3 were biologically tested according to GB/T16886.1, and the homogenization process was controlled to a temperature below 25 ℃ to avoid damage to the triple helix structure due to high temperature, the results are shown in Table 7.

TABLE 7 biocompatibility

As can be seen from Table 7, the materials prepared in examples 1 to 9 of the present application were determined to have no cytotoxicity, no acute systemic toxicity, no skin sensitization, oral viscosity stimulation index of 0.0, negative in aseptic detection, and bacterial endotoxin of less than 0.5EU/g.

EXAMPLE 15 biodegradability

1g of the sample was dried at 105 ℃ to a constant weight, and the weight m after measurement of the constant weight ₁ Adding into penicillin bottle containing 50ml of normal saline, sealing, incubating at 37 deg.C, taking out sample at set time, filtering, taking out residue, keeping constant weight (105 deg.C), measuring the weight of residue, and recording as m ₂ Then degradation rate = (m) ₁ -m ₂ )/m ₁ The results are shown in Table 8.

TABLE 8 biodegradability

As can be seen from Table 8, the degradation rate of the materials of examples 1 to 9 of the present application is slow and stable, and the degradation rate is suitable for the growth of bone tissues, thereby facilitating the formation of new bones, and simultaneously ensuring the structural stability of the biomaterial and enhancing the supporting effect. The collagen in the repair material of comparative example 1, which was actually spongy after lyophilization, degraded faster than the self-assembled fibers in example 1; while comparative example 3 is gelatin, which is a collagen degradation product, whose structure has been destroyed, degrading more rapidly; comparative example 2 because the collagen and mineral substance mixing mode was direct mechanical mixing in the course of making, make two-phase mixing through stirring, because the collagen is fibrous structure already before mixing, easy to take place the reunion in the mixing process, make collagen and mineral substance content deviation in the freeze-dried sample great, cause the product degradation rate to fluctuate greatly, stability is not enough.

Although embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A bone tissue defect repair material is characterized by comprising collagen fibers and bone powder, wherein the mass ratio of the collagen fibers to the bone powder is 1;

the self-assembly process is as follows: uniformly dispersing collagen in an acid or alkali solution to form a semitransparent collagen suspension, adding bone meal, stirring, adjusting the pH value to an isoelectric point, and self-assembling to obtain a mixed suspension of white short fibers and the bone meal; wherein the stirring speed is 1-12krpm, the pH is adjusted to 4-7.8, and the temperature is 10-25 ℃;

the particle size of the bone meal is 0.1-3.0 mm, the porosity is 50-90%, and the pore size distribution is 0.01-1500 mu m;

the length of the collagen fiber in the repair material is 0.1-5 mm.

2. The bone tissue defect repair material according to claim 1, wherein the mass ratio of the fibrous collagen to the bone powder is 3.

3. The material for repairing bone tissue defect according to claim 1, wherein said collagen fiber is formed by self-assembly of type I collagen, and said bone powder is selected from the group consisting of decellularized bone and synthetic bone.

4. The bone tissue defect repair material according to claim 3, wherein the decellularized bone is selected from one of a allogeneic bone, decellularized bovine bone or decellularized porcine bone, and the synthetic bone is selected from one of HA, TCP or multiphase composite ceramic.

5. The bone tissue defect repair material according to claim 4, wherein the multiphase composite ceramic is HA-TCP two-phase composite ceramic, and the mass ratio of HA to TCP in the HA-TCP two-phase composite ceramic is 3.

6. The method for preparing a bone tissue defect repair material according to claim 1, comprising:

a semitransparent collagen suspension formed by uniformly dispersing collagen in an acid or alkali solution, wherein the fibrous collagen keeps an intact triple-helical structure;

adding bone meal into the semitransparent collagen suspension, stirring, adjusting the pH value to an isoelectric point, and self-assembling to obtain a mixed suspension of white short fibers and the bone meal; the stirring speed in the self-assembly process is 1-12 krpm, the isoelectric point is pH 4-7.8, and the temperature is 10-25 ℃;

and (3) freezing and drying the concentrate after shaping.

7. The method for preparing a bone tissue defect repair material according to claim 6, wherein the acid solution is one or more selected from hydrochloric acid, acetic acid, lactic acid solution or citric acid; the alkali solution is selected from NaOH and NaHCO ₃ 、Na ₂ CO ₃ Or KOH solution.