CN113694254A - Bone repair material, preparation method and application thereof - Google Patents
Bone repair material, preparation method and application thereof Download PDFInfo
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- CN113694254A CN113694254A CN202111037566.8A CN202111037566A CN113694254A CN 113694254 A CN113694254 A CN 113694254A CN 202111037566 A CN202111037566 A CN 202111037566A CN 113694254 A CN113694254 A CN 113694254A
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- collagen
- bone
- repair material
- bone repair
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Abstract
The disclosure belongs to the field of biomedical materials, and particularly relates to a bone repair material, a preparation method of the bone repair material and application of the bone repair material. According to the preparation method of the bone repair material, the hydroxyapatite particles are uniformly wrapped inside the collagen fiber three-dimensional network by utilizing the self-organization of the hydroxyapatite particles and the collagen, so that the bone repair material with a porous structure and plastic characteristics is prepared. The bone repair material has high mechanical strength and can not disperse and break in the using and transporting processes; the bone repairing material has good plasticity, can be cut randomly according to the shape of a defect area, is softened when meeting water and has high elasticity, is suitable for filling bone defects at any position, and plays the functions of filling bones and guiding bone tissue regeneration.
Description
Technical Field
The disclosure belongs to the field of biomedical materials, and particularly relates to a bone repair material, a preparation method of the bone repair material and application of the bone repair material.
Background
Bone defects or bone injuries can be caused by factors such as trauma, body aging, tumors and bone diseases. In orthopaedic clinical procedures, the introduction of tissue regeneration by bone grafting is a common method of bone repair. In oral clinic, in order to deal with alveolar bone defect or insufficient alveolar bone estimation caused by tooth extraction, trauma and the like, bone in an alveolar region needs to be subjected to reparative filling before oral implantation.
In the bone defect area, the bone repair bracket with bioactivity is filled in the bone defect area, and the bioactive material can regulate and control the microenvironment around the bone tissue and controllably induce the vital activity of cells, so that the bone repair capability is greatly improved, and the bone repair bracket is the main direction for the development of the bone repair material. The filling material has the effects of promoting cell adhesion and proliferation, promoting angiogenesis, recruiting stem cells and promoting osteogenic differentiation, and further accelerating the repair and regeneration of bones in the defect area.
Currently, bone repair materials for clinical transplantation mainly include autologous bones, allogeneic bones and artificial bone repair materials. Since autologous bone sources are limited and the obtained materials can cause secondary operation injuries to patients, the autologous bone transplantation operation cannot meet the requirements of bone defect patients with large bone mass requirements. Allogeneic bone transplantation exposes the patient to secondary risks associated with immune rejection and the spread of serious disease. Therefore, the search for new bone repair materials to overcome the defects of the traditional materials is a hot spot of current research in the field of bone repair.
Citation 2 discloses a moldable bone repair material, the preparation method of which comprises the following steps: (1) completely soaking the ordered inorganic porous material in a macromolecular organic matter (collagen) solution prepared from 0.5M acetic acid, wherein the concentration of the macromolecular organic matter is 0.5-5 wt%, and stirring for 2h at 40 ℃ to prepare a suspension; (2) adjusting the pH value of the suspension to 5.5-6.5, adding an EDC/NHS cross-linking agent, wherein the mass ratio of EDC to collagen is 2:1, and the molar ratio of EDC to NHS is 4:1, fully stirring, and standing at room temperature for 24h to obtain a cross-linked product; (3) and (3) washing the crosslinked product for multiple times by adopting phosphate buffer solution, then freezing the crosslinked product for 48 hours in an environment of 20 ℃ below zero, and finally freeze-drying the crosslinked product for 24 hours under the vacuum condition of 54 ℃ below zero to obtain the plastic bone repair material. Although the bone repair material prepared by the method has plasticity, a cross-linking agent is required to be used in the preparation process, certain cytotoxicity exists, and the biological safety of the bone repair material is reduced.
Cited documents:
cited document 1: CN109954167A
Cited document 2: CN110124110A
Disclosure of Invention
Problems to be solved by the invention
In view of the problems in the prior art, for example, in the bone repair material prepared by the prior method, hydroxyapatite cannot be uniformly distributed in the collagen scaffold; the existing bone repair material is powdery and has extremely low mechanical strength and material plasticity; and the preparation process of the bone repair material needs to use a cross-linking agent, resulting in the defect of low biological safety. To this end, the present disclosure provides a method for preparing a bone repair material, the bone repair material prepared by the method, the hydroxyapatite of which can be uniformly loaded in a collagen scaffold having a three-dimensional porous structure. The bone repair material has excellent mechanical property, biocompatibility, plasticity and degradation rate matched with tissues, is suitable for filling a bone defect position, is used for retaining alveolar sites, filling alveolar bone defects or filling bone defects at any non-bearing position, and plays the effects of inducing bone repair and reconstruction.
Means for solving the problems
In a first aspect, the present disclosure provides a method for preparing a bone repair material, wherein the method comprises the following steps:
a dissolving step: preparing a solution comprising collagen at a first temperature;
a dispersing step: dispersing hydroxyapatite in the dissolved solution, and uniformly stirring at a first temperature to obtain a mixed solution;
an incubation step: placing the mixed solution in an isotropic pressure environment, and incubating at a second temperature to form a collagen scaffold; the hydroxyapatite particles are loaded in the collagen scaffold and/or on the surface of the collagen scaffold to complete the self-assembly of the hydroxyapatite and the collagen;
a freeze-drying step: carrying out freeze drying treatment on the product of the incubation step to obtain the bone repair material;
wherein the first temperature is 2-10 ℃, preferably 4 ℃; the second temperature is 20-40 deg.C, preferably 37 deg.C.
In some embodiments, the method of making according to the present disclosure, wherein the dissolving step comprises:
dissolving collagen in a pre-cooled phosphate buffer at a first temperature to obtain a collagen-containing solution; wherein the concentration of collagen in the dissolving solution is 0.5-2% (w/v), preferably 1% (w/v).
In some embodiments, the method of preparation according to the present disclosure, wherein the collagen is derived from small intestine submucosa, preferably porcine small intestine submucosa.
In some embodiments, the method of making according to the present disclosure, wherein the incubating step comprises:
and placing the mixed solution into a pre-cooled mold, applying isotropic pressure to the mold for briquetting, and incubating at a second temperature for 10-60min to complete the self-assembly of the hydroxyapatite and the collagen.
In some embodiments, the method of manufacturing according to the present disclosure, wherein the lyophilizing step comprises: placing the bone repair material into a cold trap of a freeze dryer for precooling for 60-120min, starting a vacuum pump after precooling, and carrying out vacuum drying, wherein the temperature of the cold trap is set to-35 ℃ to-45 ℃, and is preferably-40 ℃.
In some embodiments, the method of preparation according to the present disclosure, wherein the collagen is a native collagen material derived from the small intestine submucosa, the native collagen material comprising type I collagen and type III collagen; based on the total mass of the natural collagen material, the mass percentage of the type I collagen is 30-35%, and the mass percentage of the type III collagen is 65-70%.
In some embodiments, the native collagen material has a residual amount of DNA less than 4ng/mg and a residual amount of α -Gal less than 1.0U/L.
In some embodiments, the method for preparing the native collagen material comprises the following steps:
taking a small intestine submucosa material, and sequentially carrying out virus inactivation treatment, degreasing treatment, cell removal treatment and enzymolysis treatment to obtain a collagen crude extract;
purifying and drying the crude collagen extraction solution to obtain a natural collagen material;
preferably, the native collagen material is a native collagen material according to the first aspect.
In some embodiments, the method of manufacturing according to the present disclosure, wherein the step of virus inactivation treatment comprises: cleaning a small intestine submucosa material, soaking the small intestine submucosa material in a virus inactivation solution, and performing virus inactivation treatment under the oscillation condition; preferably, the volume ratio of the small intestine submucosa material to the virus inactivation solution is (0.5-5): 30; preferably, the virus inactivation solution is an aqueous solution comprising (0.5-5)% (v/v) peroxyacetic acid and (20-25)% (v/v) ethanol; preferably, the time of the virus inactivation treatment is (1-5) h.
In some embodiments, the method of manufacturing according to the present disclosure, wherein the step of degreasing comprises: soaking the virus-inactivated small intestine submucosa material in a degreasing solution, and performing degreasing treatment under the ultrasonic condition; preferably, the volume ratio of the small intestine submucosa material to the degreasing solution is (0.1-1): 2; preferably, the degreasing solution is 1-2 wt% alkaline lipase solution; preferably, the degreasing solution has a pH of 7-10; preferably, the small intestine submucosa material is soaked in the degreasing fluid, the degreasing fluid is replaced after ultrasonic treatment for 30-60min, and the operation is repeated for more than 3 times.
In some embodiments, the method of manufacturing according to the present disclosure, wherein the step of decellularizing comprises: soaking the degreased small intestine submucosa material in a cell removing solution, and performing cell removing treatment under the ultrasonic condition; preferably, the volume ratio of the small intestine submucosa material to the decellularization solution is (0.5-5): 30; preferably, the decellularization solution is a phosphate buffer comprising 0.02-0.05 wt% trypsin and 0.1-0.4 wt% EDTA; preferably, the time of the decellularization treatment is 30min, and then the decellularized small intestine submucosa material is obtained by washing and drying.
In some embodiments, the preparation method according to the present disclosure, wherein the step of performing enzymatic treatment comprises:
taking a small intestine submucosa material without cells, crushing the small intestine submucosa material, and soaking the small intestine submucosa material in an acid solution;
crushing and stirring the soaked small intestine submucosa material by adopting crushing equipment, adding pepsin, performing enzymolysis treatment, centrifuging, and removing precipitates to obtain a collagen crude extract;
preferably, the volume ratio of the small intestine submucosa material to the acidic solution is (0.5-5): 30; preferably, the acid solution is (0.1-1) mol/L acetic acid solution, the time for soaking the small intestine submucosa material in the acid solution is 12h, and the temperature equipment is 4-10 ℃; preferably, the mass ratio of the small intestine submucosa material to the pepsin is (8-12) to 1; preferably, the enzymolysis treatment is carried out for 48h to 96h under the stirring condition.
In some embodiments, the method of manufacturing according to the present disclosure, wherein the step of purifying comprises:
filtering the crude collagen extraction solution for one time, and then performing salting-out treatment to obtain precipitate particles; dissolving the precipitate particles in an acidic solution, washing and dissolving for the second time, and performing membrane separation, filtration and concentration to obtain a collagen concentrated solution containing natural collagen materials.
In a second aspect, the present disclosure provides a bone repair material, wherein the bone repair material comprises:
a collagen scaffold having a three-dimensional porous structure;
hydroxyapatite particles loaded inside, and/or on the surface of, the collagen scaffold; preferably, the hydroxyapatite particles are uniformly loaded on the surface and/or inside the collagen scaffold; preferably, the bone repair material is prepared by the method according to the first aspect.
In some embodiments, the bone repair material according to the present disclosure, wherein the collagen scaffold has a mass fraction of 3.3% to 10% and the hydroxyapatite particles have a mass fraction of 90% to 96.7% based on the total mass of the bone repair material.
In some embodiments, the bone repair material according to the present disclosure, wherein the collagen scaffold has a porosity of not less than 80%, and the hydroxyapatite particles have a particle size of 100-150 μm.
In some embodiments, the bone repair material according to the present disclosure, wherein the collagen scaffold is formed from collagen derived from small intestine submucosa.
In some embodiments, the use of a bone repair material according to the second aspect of the disclosure, or a bone repair material prepared according to the method of the first aspect, as or in the preparation of a bone filler material as one or more of: reserving alveolar sites after tooth extraction, filling alveolar bone defects, or filling bone defects at any non-bearing positions;
optionally, the bone defect at the non-weight bearing location is selected from one or more of: bone defects of maxillofacial surgery, bone defects of dental surgery, skull defects and various closed fracture bone defects of four limbs.
ADVANTAGEOUS EFFECTS OF INVENTION
In some embodiments, the preparation method of the bone repair material provided by the present disclosure prepares a bone filling material having a porous structure and plastic characteristics by compounding hydroxyapatite particles with collagen derived from porcine small intestine submucosa. The method adopts the self-assembly characteristic of collagen, and uniformly wraps hydroxyapatite particles in the collagen fiber three-dimensional network, so that the material cannot be dispersed and broken in the using and transporting processes. The preparation process does not add any chemical cross-linking agent or adopt high-temperature cross-linking, retains the biological activity of collagen and the bone guiding property of hydroxyapatite, and the addition of the collagen enhances the biological activity of the bone filling material, and compared with the bone filling material which only takes the hydroxyapatite as the raw material, the repair effect is more obvious. In addition, the collagen can guide and promote the adhesion and proliferation of cells, accelerate the regeneration of blood vessels in the defect area and provide sufficient nutrient supply for the regeneration of bones in the defect area. Meanwhile, the self-assembled bone repair material has good hydrophilicity and can be well attached to a tissue defect area by the collagen. Finally, the hydroxyapatite particles used are stable in performance and are slowly absorbed by the human body, mainly through osseointegration, but do not cause changes in the pH of the surrounding environment, relative to the degradable calcium phosphate materials.
And uniformly mixing the collagen dissolving solution and the hydroxyapatite, and then carrying out an incubation step to enable the hydroxyapatite to be uniformly loaded in and/or on the surface of the collagen scaffold. The bone repair material obtained after freeze-drying has high mechanical strength and can not disperse and break in the using and transporting processes. The bone repair material has good plasticity, can obtain blocky bone repair materials, can be cut randomly according to the shape of a defect area, becomes soft and has elasticity when meeting water, is suitable for filling bone defects of any site and retaining alveolar sites; in addition, the bone repair material has good hydrophilicity and high bioactivity, has good bone guiding and bone inducing capabilities, can be tightly attached to a tissue defect part, and plays the functions of filling bones and guiding bone tissue regeneration.
In some embodiments, the present disclosure provides a method for preparing a bone repair material, which does not require the use of a cross-linking agent and does not require high-temperature cross-linking during the preparation process, retains high bioactivity of collagen, and has high biosafety.
In some embodiments, the three-dimensional porous structure of the collagen scaffold provided by the present disclosure is suitable for adhesion and proliferation of cells, accelerates angiogenesis in the defect area, provides sufficient nutrient supply for bone regeneration in the defect area, and promotes bone repair. The hydroxyapatite particles are uniformly loaded on the surface and/or inside the collagen scaffold, and the bone repair material has better mechanical property, high bioactivity and histocompatibility.
Drawings
FIG. 1 shows a pictorial view of an SIS/HA composite bone repair material;
FIG. 2 shows a cross-sectional view of the SIS/HA composite bone repair material after cutting;
FIG. 3 shows the hydrophilicity test results of the SIS/HA composite bone repair material, wherein a is a picture before water absorption of the SIS/HA composite bone repair material, b is a picture after water absorption of the SIS/HA composite bone repair material, and c is a picture after excessive water absorption of the SIS/HA composite bone repair material;
FIG. 4 shows an electron microscope photograph of the internal morphology of the SIS/HA composite bone repair material, wherein a-c in FIG. 4 sequentially show electron microscope photographs with scales of 1.00mm, 300 μm and 200 μm;
FIG. 5 shows a compression performance analysis of SIS/HA bone repair material;
FIG. 6 shows the results of the leachate cytotoxicity assays, where SIS/HA represents the composite bone repair material, HA represents the hydroxyapatite raw material, and control represents the blank.
Detailed Description
Various exemplary embodiments, features and aspects of the invention will be described in detail below. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.
All units used in the specification are international standard units unless otherwise stated, and numerical values and numerical ranges appearing in the present invention should be understood to include systematic errors inevitable in industrial production.
In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.
In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.
In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.
In the present specification, "v/v" represents a volume percentage content, "wt%" represents a mass percentage content, and "w/v" represents a "g/ml" concentration.
In addition, in the present specification, the "water" includes any feasible water that can be used, such as deionized water, distilled water, ion-exchanged water, double distilled water, high purity water, and purified water.
In the present specification, when "normal temperature" or "room temperature" is used, the temperature may be 10 to 40 ℃.
Preparation method of bone repair material
A first aspect of the present disclosure provides a method of preparing a bone repair material, comprising the steps of:
a dissolving step: preparing a solution comprising collagen at a first temperature;
a dispersing step: dispersing hydroxyapatite in the dissolved solution, and uniformly stirring at a first temperature to obtain a mixed solution;
an incubation step: placing the mixed solution in an isotropic pressure environment, and incubating at a second temperature to form a collagen scaffold; the hydroxyapatite particles are loaded in the collagen scaffold and/or on the surface of the collagen scaffold to complete the self-assembly of the hydroxyapatite and the collagen;
a freeze-drying step: carrying out freeze drying treatment on the product of the incubation step to obtain the bone repair material;
wherein the first temperature is 2-10 ℃, illustratively 3 ℃, 4 ℃, 5 ℃, 6 ℃, 8 ℃, 9 ℃ and the like; preferably, the first temperature is 4 ℃. The second temperature is 20-40 deg.C, illustratively 22 deg.C, 24 deg.C, 26 deg.C, 28 deg.C, 30 deg.C, 32 deg.C, 34 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, etc.; preferably, the second temperature is 37 ℃.
According to the preparation method, the collagen and the hydroxyapatite are uniformly mixed in a state that the collagen is not self-assembled by performing the dissolving step and the dispersing step at the first temperature, so that the hydroxyapatite is prevented from being unevenly distributed on the collagen scaffold, the bone repair material has high mechanical compression strength, and a stable and reliable environment is provided for bone regeneration. The mixed solution is placed in an isotropic pressure environment for briquetting, and finally, the massive bone repair material is obtained, has good plasticity and hydrophilicity, is suitable for being filled in bone defect areas at any non-bearing positions, is well attached to tissue defect areas, plays a role in bone filling, and can promote the repair and regeneration of bone tissues.
In some embodiments, the dissolving step comprises: dissolving collagen in a pre-cooled phosphate buffer at a first temperature to obtain a collagen-containing solution; wherein the concentration of collagen in the dissolution solution is 0.5% -2% (w/v), and illustratively, the concentration of collagen in the dissolution solution is 0.8% (w/v), 1% (w/v), 1.2% (w/v), 1.5% (w/v), 1.8% (w/v), and the like. Preferably, the concentration of collagen in the dissolution solution is 1% (w/v). In some alternative embodiments, the collagen is solubilized at low temperatures of 4 ℃ using 4-10 ℃ pre-chilled PBS buffer (1 ×) to give a collagen solution with a concentration of 0.5% -2% (w/v).
Dissolving collagen at the first temperature can avoid self-assembly of collagen before mixing with hydroxyapatite uniformly, and avoid uneven distribution of hydroxyapatite in the collagen scaffold. The concentration of collagen in the dissolving solution is 0.5-2% (w/v), so that the collagen solution has sufficient fluidity and can be fully and uniformly mixed with the hydroxyapatite; meanwhile, the collagen material can fully wrap hydroxyapatite particles after freeze-drying, and the composite structure formed by the collagen and the hydroxyapatite ensures the strength of the bone repair material. The bone repair material has higher structural strength in a dry state, and a user can cut and shape the bone repair material according to the shape of the defect to be repaired in the using process, so that the specific shape filling is realized. In addition, after the bone powder filling material is filled into a defect position, even if the bone powder filling material is soaked by liquid, the shape of the block mass after cutting and shaping cannot collapse, the filling effect at the defect position is ensured, and the bone powder cannot flow along with the liquid and run off from the filling position.
In some preferred embodiments, the collagen is derived from Small Intestinal Submucosa (SIS), more preferably porcine Small Intestinal Submucosa. The SIS collagen has high content of type III collagen, and the bone repair material prepared from the SIS collagen has improved bioactivity and bone performance. SIS collagen and hydroxyapatite (Ca)10(PO4)6(OH)2HA) is blended to prepare the SIS/HA composite bone repair material, HAs good biocompatibility, osteoconductivity and osteoinduction, and provides good cell proliferation and differentiation environment for bone repair.
In some embodiments, the dispersing step comprises: dispersing hydroxyapatite into the dissolving solution according to the mass ratio of collagen to hydroxyapatite of 1:9 to 1:29, and uniformly stirring at a first temperature to obtain a mixed solution. Illustratively, the mass ratio of collagen to hydroxyapatite is 1:10, 1:12, 1: 13. 1:14, 1:15, 1:18, 1:20, 1:22, 1:24, 1:26, and so forth. The hydroxyapatite and the collagen are stirred and mixed at a first temperature, so that the hydroxyapatite and the collagen are uniformly distributed in the mixed solution under the state of not self-assembling.
In some embodiments, the incubating step comprises: and placing the mixed solution into a pre-cooled mold, applying isotropic pressure to the mold for briquetting, shaping the compound, and incubating at a second temperature for 10-60 minutes to complete the self-assembly of the collagen and the hydroxyapatite. And incubating at a second temperature to enable collagen to self-assemble to form a three-dimensional porous scaffold with a three-dimensional porous structure, and uniformly distributing hydroxyapatite in the mixed solution in the interior and/or on the surface of the scaffold to obtain the bone repair material with high mechanical compression performance, so that the requirements of transportation and use are met. By briquetting, a massive bone repair material can be obtained, which has good plasticity, is suitable for specific filling of a defect region, and does not undergo morphological collapse when wetted by liquid at a defect position.
Further, in the incubation step, no cross-linking agent is added to the mixed solution. The preparation method disclosed by the invention can be used for preparing the self-assembled three-dimensional porous bone repair material without using a cross-linking agent, so that toxic and side effects caused by using the cross-linking agent are avoided.
Furthermore, in the incubation step, a high-temperature crosslinking mode is not adopted, so that the high biological activity of the collagen is kept.
In some embodiments, the lyophilizing step comprises: placing the bone repairing material into a cold trap of a freeze dryer for precooling for 60-120min, starting a vacuum pump after precooling, and carrying out vacuum drying, wherein the temperature of the cold trap is set to-35 ℃ to-45 ℃, such as-37 ℃, 38 ℃, 40 ℃, 42 ℃ and 44 ℃. Preferably, the temperature of freeze-drying is-40 ℃. The bone repair material in a dry state is obtained through the freeze-drying step, has enhanced structural strength, and is not dispersed or broken in the transportation and use processes.
According to the preparation method provided by the disclosure, collagen and hydroxyapatite are used as raw materials, and the bone repair material with bionic natural bone composition and structure can be prepared through interaction of the steps, wherein the hydroxyapatite is uniformly loaded in the collagen scaffold, and has high porosity, excellent mechanical property, good plasticity, hydrophilicity and biocompatibility. The bone repair material has a biodegradation rate matched with tissues, can be slowly absorbed by a human body through osseointegration, but does not cause the change of the pH of the surrounding environment.
Bone repair material
A second aspect of the present disclosure provides a bone repair material comprising:
a collagen scaffold having a three-dimensional porous structure;
hydroxyapatite particles loaded inside and/or on the surface of the collagen scaffold.
The bone repair material disclosed by the disclosure is formed by assembling hydroxyapatite particles and a collagen scaffold, and the three-dimensional porous structure of the collagen scaffold is suitable for cell adhesion and proliferation, so that the repair and reconstruction of bone tissues are promoted. The bone repair material simulates the structure and composition of natural bone, has good biocompatibility, proper mechanical property and biodegradation rate matched with tissues, and is suitable for being filled in bone defect areas to promote the induction and regeneration of bone.
In some preferred embodiments, the bone repair material is prepared by a method according to the first aspect. The bone repair material is blocky, has excellent plasticity and hydrophilicity, and can be well attached to a tissue defect area.
In some preferred embodiments, hydroxyapatite particles are uniformly loaded in and/or on the surface of the collagen scaffold, and the bone repair material has good mechanical compression performance, so that the requirements of bone filling and transportation are met.
In some embodiments, the collagen scaffold is formed from collagen derived from small intestine submucosa, preferably porcine small intestine submucosa. The collagen scaffold assembled by the SIS collagen has higher bioactivity and contributes to bone property.
In some embodiments, the collagen scaffold has a mass fraction of 3.3 to 10% and the hydroxyapatite particles have a mass fraction of 90 to 96.7%, based on the total mass of the bone repair material; illustratively, the collagen scaffold has a mass fraction of 4%, 4.5%, 5%, 5.5%, 6%, 6.3%, 6.7%, 7%, 7.3%, 7.5%, 7.8%, 8%, 8.2%, 8.4%, 8.6%, 8.8%, 9%, 9.3%, 9.5%, etc., and the hydroxyapatite particles have a mass fraction of 91%, 91.5%, 92%, 92.5%, 93%, 93.3%, 93.7%, 94%, 95%, 96%, etc. Preferably, the mass fraction of the collagen scaffold is 6.7%, and the mass fraction of the hydroxyapatite particles is 93.3%.
In some embodiments, the bone repair material is prepared by mixing the collagen scaffold and the hydroxyapatite particles in a mass ratio of 1:9 to 1: 29; preferably, the mass ratio of the collagen scaffold to the hydroxyapatite particles is from 1:13 to 1: 15; more preferably, the mass ratio of the collagen scaffold to the hydroxyapatite particles is 1: 14.
In some embodiments, the collagen scaffold has a porosity of greater than 80%; the particle size of the hydroxyapatite particles is 100-150 mu m.
Collagen dissolving solution
The collagen lysates used in the present disclosure are taken from natural collagen materials, including type I collagen and type III collagen; based on the total mass of the natural collagen material, the content of the type I collagen is 30-35%, and the content of the type III collagen is 65-70%;
the natural collagen material is derived from small intestine submucosa, preferably pig small intestine submucosa.
The natural collagen material disclosed by the invention is obtained by taking Small Intestinal Submucosa (SIS) as a raw material, the content of III-type collagen in the extracted collagen is higher, a natural, safe and low-immunogenicity collagen source is provided for supplementing the loss of collagen, and the natural collagen material is suitable for serving as a collagen scaffold and is self-assembled with hydroxyapatite to form an orthopedic repair material.
In some preferred embodiments, the native collagen material is derived from porcine small intestine submucosa, which contains a high level of type III collagen.
In some embodiments, the natural collagen material provided by the present disclosure has a low content of immunogenic substances, wherein the residual amount of DNA is less than 4ng/mg, and the detection of α -Gal is performed by an enzyme-linked immunosorbent assay, and the residual amount thereof is less than 1.0U/L of the detection limit of the α -galactosidase detection kit. The natural collagen material has low content of immunogenic substances and high biological safety, and is suitable for being implanted into tissues.
The preparation method of the natural collagen solution comprises the following steps:
taking a small intestine submucosa material, and sequentially carrying out virus inactivation treatment, degreasing treatment, cell removal treatment and enzymolysis treatment to obtain a collagen crude extract;
and purifying and drying the crude collagen extraction solution to obtain a natural collagen material.
The collagen solution may be prepared by directly using the purified solution or by re-dissolving the natural collagen material.
The preparation method disclosed by the invention can effectively remove intracellular components such as nucleic acid fragments, cell membranes, cell nucleus fragments and the like and reduce the residues of immune source substances through degreasing and decellularization treatment of the small intestine submucosa material. Compared with the traditional tissue collagen extraction method, the preparation method disclosed by the invention can effectively simplify the purification treatment steps of collagen, and can be used for preparing the natural collagen material with high type III collagen content and high biological safety. Compared with the method for preparing the type III collagen by genetic engineering, the preparation method disclosed by the invention has the advantages of low cost, simple steps, easiness in operation and the like.
In some embodiments, using the preparation methods of the present disclosure, a natural collagen material can be prepared having a collagen type I content of 30-35% and a collagen type III content of 65-70% by total mass.
In some embodiments, the small intestine submucosa material is the intermediate small intestine submucosa material that is left after the mucosal and fascia layers of the small intestine have been removed. The small intestine submucosa material is washed with clear water and used for subsequent treatment.
In some embodiments, the step of virus inactivation treatment comprises: the small intestine submucosa material is cleaned and then soaked in a virus inactivation solution, and virus inactivation treatment is carried out under the oscillation condition.
For the virus inactivation solution, it is an aqueous solution comprising (0.5-5)% (v/v) peroxyacetic acid and (20-25)% (v/v) ethanol. Illustratively, the amount of peroxyacetic acid in the virus inactivation solution is 0.8% (v/v), 1% (v/v), 1.5% (v/v), 2% (v/v), 2.5% (v/v), 3% (v/v), 3.5% (v/v), 4% (v/v), 4.5% (v/v), etc.; the content of ethanol in the virus-inactivating solution is 21% (v/v), 22% (v/v), 23% (v/v), 24% (v/v), or the like. In some preferred embodiments, the virus inactivation solution is an aqueous solution comprising 1% (v/v) peroxyacetic acid and 24% (v/v) ethanol.
The ratio of the volume of the small intestine submucosa material to the volume of the virus inactivation solution is (0.1-1):2, e.g., 0.2:2, 0.4:2, 0.6:2, 0.8:2, etc. In some preferred embodiments, the volume ratio of small intestine submucosa material to virus inactivation solution is 1: 5. Soaking the small intestine submucosa material in a virus inactivation solution, and soaking under the oscillation condition. The small intestine submucosa material is soaked in the virus inactivating solution for 1-5h, preferably for 2h, and the shaking speed is 40 r/min. The soaked small intestine submucosa material is washed with water. The oscillation can be achieved by means of a shaker, magnetic stirrer, or the like.
By the virus inactivation treatment, virus components in the small intestine submucosa material can be effectively inactivated, the content of bacterial endotoxin can be further reduced, and the biological safety of the natural collagen material is ensured.
In some embodiments, the step of degreasing comprises: and (3) soaking the virus-inactivated small intestine submucosa material in a degreasing solution, and performing degreasing treatment under the ultrasonic condition.
Further, the degreasing solution is 1-2 wt% alkaline lipase solution. Illustratively, the concentration of the alkaline lipase solution is 1 wt%, 1.2 wt%, 1.4 wt%, 1.5 wt%, 1.7 wt%, 2 wt%, etc. In some preferred embodiments, the pH of the degreasing solution is 7-10, e.g., pH 7, 8, 9, 10, etc. Exemplary small intestine submucosa material to degreasing solution volume ratios are 0.1:2, 0.2:2, 0.5:2, 0.8:2, 1:2, and so forth, in terms of a small intestine submucosa material to degreasing solution volume ratio of (0.1-1): 2. Preferably, the volume ratio of the small intestine submucosa material to the degreasing solution is 1:10, the small intestine submucosa material is soaked in the degreasing solution, and the degreasing solution is replaced after degreasing treatment is carried out for 30-60min under the ultrasonic condition; the procedure was repeated 3 more times and then the small intestine submucosa material was washed with water. Wherein the ultrasonic frequency is 30kHz-50kHz, preferably 40 kHz; the power was 5 kw.
Through degreasing treatment, non-collagen impurity components mixed in the crude collagen extracting solution can be effectively reduced, so that the subsequent purification treatment process is simplified, and the loss of collagen is avoided. Compared with the existing treatment mode of placing tissues in water for ultrasonic cleaning, the alkaline lipase solution with the concentration of 2 wt% is selected in the method, so that the unwanted impurity components such as lipid can be more effectively removed, and the loss of collagen components in the extraction process is reduced.
In some embodiments, the step of decellularizing comprises: and (3) soaking the degreased small intestine submucosa material in a cell removing solution, and performing cell removing treatment under the ultrasonic condition.
For the decellularization solution, a phosphate buffer containing 0.02-0.05 wt% trypsin and 0.1-0.4 wt% EDTA. Illustratively, the content of trypsin in the phosphate buffer is 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, etc., and the content of EDTA is 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, etc. In some preferred embodiments, the phosphate buffer contains 0.02 wt% trypsin and 0.1 wt% EDTA. Soaking the small intestine submucosa material in the cell removing solution according to the volume ratio of the small intestine submucosa material to the cell removing solution of (0.5-5) to 30, and soaking for 30min under the ultrasonic condition. Further, the small intestine submucosa material and the cell removing solution are soaked in an ultrasonic cleaning machine for 30min according to the volume ratio of 1: 30. Wherein the ultrasonic frequency is 20kHz-30kHz, preferably 28 kHz; the power was 5 kw.
After the ultrasound treatment, the small intestine submucosa material is washed and dried. In some more specific embodiments, the drying is carried out under conditions in which the small intestine submucosa material is dried at a temperature of 37 ℃ for 24 hours.
The conditions of the cell removing treatment in the method are mild, so that the immunogenic components such as nucleic acid, cell membranes, cell nucleus fragments and the like can be sufficiently removed, the immunogenicity of the natural collagen material is reduced, the natural collagen material is suitable for being applied to a bone defect area, and the loss of the collagen component is avoided.
In the conventional collagen extraction process, after a crude collagen extraction solution is obtained, it is necessary to repeat a purification operation of salting out and dissolving to remove impurity components such as nucleic acid mixed in collagen. This process results in loss of collagen and, due to the use of large amounts of neutral salts, requires washing or dialysis at a later stage, is time consuming and complicated to operate. In the method, the immune source substances such as fat, nucleic acid, alpha-Gal and the like can be reduced to the minimum by combining degreasing and decellularization treatment, so that the purification process of the crude collagen solution can be completed by only once salting out and microfiltration membrane filtration.
In some embodiments, the step of enzymatically treating comprises: taking a small intestine submucosa material without cells, crushing the small intestine submucosa material, and soaking the small intestine submucosa material in an acid solution; and (3) crushing and stirring the soaked small intestine submucosa material by adopting crushing equipment, adding pepsin, performing enzymolysis treatment, centrifuging, and removing precipitates to obtain a collagen crude extract solution.
Further, the acidic solution is an acetic acid solution of 0.1 to 1mol/L, and illustratively, the concentration of the acetic acid solution is 0.2mol/L, 0.3mol/L, 0.5mol/L, 0.7mol/L, 0.8mol/L, or the like. Preferably, the concentration of the acetic acid solution is 0.5 mol/L. The dried small intestine submucosa material is cut into pieces and then mixed with the acidic solution at a volume ratio of small intestine submucosa material to acidic solution of (0.5-5):30, e.g., 0.8:30, 1:30, 2:30, 3:30, 4:30, etc. In some preferred embodiments, the small intestine submucosa material is soaked in an acidic solution in a volume ratio of 1: 30. In some preferred embodiments, the small intestine submucosa material is soaked in an acidic solution for 12 hours at a low temperature of 4-10 ℃ to facilitate the solubilization of collagen.
For the enzymolysis treatment, the soaked small intestine submucosa material is pulverized and stirred at low temperature for 5min by a pulverizer, then pepsin is added into the acidic solution according to the mass ratio of 1 (8-12) of the pepsin to the small intestine submucosa material, and the mixture is mechanically stirred at low temperature (4-10 ℃) for 48-96h, so that the collagen in the small intestine submucosa material is fully dissolved, and the extraction efficiency of type III collagen and type I collagen is improved. In some preferred embodiments, the mass ratio of pepsin to small intestine submucosa material is 1: 10; after adding pepsin into the acidic solution, the time of low-temperature mechanical stirring is 72 h.
After the enzymolysis treatment, the mixed solution is centrifuged, and the obtained supernatant is the collagen crude extraction solution dissolved with the collagen.
In some embodiments, the step of purifying comprises: filtering the crude collagen extraction solution for one time, and then performing salting-out treatment to obtain precipitate particles; dissolving the precipitate particles in an acidic solution, washing and dissolving for the second time, and performing membrane separation, filtration and concentration to obtain a collagen concentrated solution containing natural collagen materials.
Further, the acidic solution is an acetic acid solution of 0.5mol/L to sufficiently dissolve the native collagen material in the precipitated particles.
For filtration, the solution is filtered using a microfiltration membrane to filter out particles in the solution. Wherein, the micro-filtration membrane aperture of the first filtration is 0.2-0.4 μm, and insoluble particles in the collagen crude extract solution can be removed by the first filtration. The aperture of the filter membrane filtered after the secondary cleaning is 0.05-0.1 μm, and the redundant micromolecular impurities can be removed through the filtering after the secondary cleaning, so that the purity of the collagen concentrated solution is improved. In the purification step in the method, only pepsin and residual non-collagen components in the material need to be removed, so that the purification of the crude collagen extraction solution can be completed only by once salting out and microporous membrane filtration, and the method has the advantages of simple steps and less loss of collagen.
Then, the collagen concentrated solution was freeze-dried to obtain a natural collagen material, and then the collagen solution was dissolved in cold PBS at a low temperature (4 ℃ C.).
Use of bone repair material
A third aspect of the present disclosure provides a bone repair material prepared by the method provided by the first aspect, or a use of the bone repair material provided by the second aspect. The bone repair material disclosed by the disclosure has good mechanical property, bioactivity and plasticity, and is suitable for filling bone defects of any shape as a bone filling material.
In some embodiments, the bone repair material is used for alveolar site preservation after tooth extraction, avoiding the absorption and degradation of alveolar bone, and stabilizing gum. In some embodiments, the bone repair material is used for filling alveolar bone defects, effectively supplementing the bone mass of alveolar bone and promoting the healing of alveolar bone. In some embodiments, the bone repair material may also be used for the filling of bone defects at any other non-weight bearing location. Exemplary, bone defects at non-weight bearing locations include, but are not limited to: bone defects of maxillofacial surgery, bone defects of dental surgery, skull defects and various closed fracture bone defects of four limbs. The bone repair material disclosed by the disclosure can be well attached to a damaged area, so that the adhesion and proliferation of cells are promoted, and the repair and reconstruction of bones at bone defect positions are promoted.
Examples
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
This example provides a method for preparing a native collagen material, comprising the steps of:
(1) cleaning pretreatment: at room temperature, the mucosal layer and fascia layer of the small intestine are scraped, the middle small intestine submucosa is left, and the material is washed by purified water for standby.
(2) Virus inactivation: preparing a virus inactivation water solution (1% (v/v) peroxyacetic acid + 24% (v/v) ethanol), soaking the cleaned small intestine submucosa material in the virus inactivation solution, wherein the volume ratio of the small intestine submucosa material to the solution is 1:30(v: v), placing the small intestine submucosa material on a shaking table, and soaking the small intestine submucosa material for 2 hours at room temperature at the speed of 40 r/min. And cleaning with purified water after soaking.
(3) Degreasing: preparing 2 wt% alkaline lipase solution, soaking the virus-killed volume in degreasing solution with the volume ratio of small intestine submucosa material to degreasing solution of 1:10(v: v), ultrasonically soaking in ultrasonic cleaning machine for 30min, replacing degreasing solution, and repeating the operation for 3 times. And (5) cleaning with purified water.
(4) And (3) cell removal: preparing a decellularized phosphate buffer solution (0.025 wt% of trypsin and 0.19 wt% of EDTA), transferring the small intestine submucosa material into a decellularized solution, ultrasonically soaking the small intestine submucosa material and the decellularized solution in an ultrasonic cleaning machine for 30min at a volume ratio of 1:30(v: v), cleaning the small intestine submucosa material by adopting purified water after ultrasonic treatment, and drying the small intestine submucosa material in an oven at 37 ℃ for 24 h.
(5) Acid soaking: cutting the dried small intestine submucosa material into about 1cm2The small intestine submucosa material after being decellularized is soaked for 12 hours by 0.5mol/L acetic acid solution at 4 ℃, and the ratio of the small intestine submucosa material to the decellularized solution is 1:30(v: v).
(6) Enzymolysis: pulverizing the soaked small intestine submucosa material with a pulverizer at low temperature, stirring for 5min, adding pepsin (the mass ratio of pepsin to small intestine submucosa material is 1:10) into the solution, and mechanically stirring at low temperature for 72 h. And after enzymolysis, centrifuging the mixed solution at 8000r/min for 10min, and taking the supernatant as a crude collagen solution.
(7) And (3) purification: the crude collagen solution is treated by a microfiltration membrane, and insoluble particles are filtered by a filter membrane with the aperture of 0.2-0.4 micron. Salting out by using neutral salt sodium chloride or ammonium sulfate, centrifuging after salting out to obtain precipitated particles, washing by using distilled water, dissolving by using 0.5mol/L acetic acid, and concentrating by using an ultrafiltration membrane with the pore diameter of 0.05-0.1 micrometer to remove redundant micromolecular impurities to obtain the high-purity collagen concentrated solution.
(8) And (3) drying: and (4) freeze-drying the high-purity collagen concentrated solution to obtain the natural collagen material.
When the volume of the small intestine submucosa material is measured, the cleaned material is placed on a stainless steel net for 1-5min and then is put into a measuring cylinder for measurement.
Example 2:
this example provides a method for preparing a bone repair material, which uses the natural collagen material prepared in example 1 to prepare a collagen dissolving solution, and includes the following steps:
(1) a dissolving step: the collagen was dissolved in cold PBS buffer at low temperature (4 ℃ C.) to obtain a solution with a collagen concentration of 1% (w/v).
(2) A dispersing step: dispersing Hydroxyapatite (HA) in a collagen dissolving solution, and uniformly dispersing and stirring at a low temperature (4 ℃) to obtain an HA/collagen mixed solution, wherein the mass ratio of collagen to hydroxyapatite is 1:29, 1:19, 1:14 and 1: 9.
(3) An incubation step: and (3) filling the HA/collagen mixed solution into a pre-cooled mould, briquetting by using an isostatic pressing device (JDP-40J, Shanghai Jingsheng scientific instruments Co., Ltd.), putting into an environment at 37 ℃ for incubation for 1h, and taking out from the mould to obtain the HA/collagen composite material.
(4) The HA/collagen composite material is put into a cold trap of a freeze dryer (FD-1A-50, Beijing Bo Yi kang laboratory instruments, Ltd.) for precooling for 60min, a vacuum pump is started after precooling for vacuum drying, and the temperature of the cold trap is set to be-40 ℃. And (5) freeze-drying to obtain the bone repair material.
Example 3:
this example provides a method for preparing a bone repair material, which uses the natural collagen material prepared in example 1 to prepare a collagen dissolving solution, and includes the following steps:
(1) a dissolving step: the collagen was dissolved in cold PBS buffer at low temperature (4 ℃ C.) to obtain a solution having a collagen concentration of 2% (w/v).
(2) A dispersing step: dispersing Hydroxyapatite (HA) in collagen dissolving solution, and uniformly dispersing and stirring at low temperature (5 ℃) to obtain HA/collagen mixed solution, wherein the mass ratio of collagen to hydroxyapatite is 1:29, 1:19, 1:14 and 1: 9.
(3) An incubation step: and (3) filling the HA/collagen mixed solution into a pre-cooled mould, briquetting by using an isostatic pressing device (JDP-40J, Shanghai Jingsheng scientific instruments Co., Ltd.), putting into an environment at 37 ℃ for incubation for 50min, and taking out from the mould to obtain the HA/collagen composite material.
(4) The HA/collagen composite material is put into a cold trap of a freeze dryer (FD-1A-50, Beijing Bo Yi kang laboratory instruments, Ltd.) for precooling for 80min, a vacuum pump is started after precooling for vacuum drying, and the temperature of the cold trap is set to be-45 ℃. And (5) freeze-drying to obtain the bone repair material.
Example 4:
this example provides a method for preparing a bone repair material, which uses the natural collagen material prepared in example 1 to prepare a collagen dissolving solution, comprising the following steps:
(1) a dissolving step: the collagen was dissolved in cold PBS buffer at low temperature (4 ℃ C.) to obtain a solution having a collagen concentration of 0.5% (w/v).
(2) A dispersing step: dispersing Hydroxyapatite (HA) in a collagen dissolving solution, and uniformly dispersing and stirring at a low temperature (4 ℃) to obtain an HA/collagen mixed solution, wherein the mass ratio of collagen to hydroxyapatite is 1:29, 1:19, 1:14 and 1: 9.
(3) An incubation step: and (3) filling the HA/collagen mixed solution into a pre-cooled mould, briquetting by using an isostatic pressing device (JDP-40J, Shanghai Jingsheng scientific instruments Co., Ltd.), putting into an environment at 37 ℃ for incubation for 30min, and taking out from the mould to obtain the HA/collagen composite material.
(4) The HA/collagen composite material is put into a cold trap of a freeze dryer (FD-1A-50, Beijing Bo Yi kang laboratory instruments, Ltd.) for precooling for 120min, a vacuum pump is started after precooling for vacuum drying, and the temperature of the cold trap is set to-35 ℃. And (5) freeze-drying to obtain the bone repair material.
FIG. 1 shows a pictorial view of an SIS/HA composite bone repair material. The bone filling and repairing material after freeze drying is a white block material, and the initial appearance of the bone filling and repairing material can be shaped through a mold according to a filling target. When the defect with the special shape is repaired, the defect with the special shape can be cut and shaped, so that the defect with the special shape is filled and the repair is completed.
FIG. 2 shows a cross-sectional view of the SIS/HA composite bone repair material after cutting.
Performance testing
The bone repair materials in the examples were tested as follows:
fig. 3 shows the hydrophilicity test results of the SIS/HA composite bone repair material, wherein a is a picture before water absorption of the SIS/HA composite bone repair material, b is a picture after water absorption of the SIS/HA composite bone repair material, and c is a picture after excessive water absorption of the SIS/HA composite bone repair material. As can be seen from the a-c diagrams of fig. 3, there is substantially no change in the morphology of the composite bone repair material after water absorption or even after excessive water absorption. The structure of the repair material is not collapsed due to water absorption. This is the role played by the collagen in the self-assembled bone repair material to maintain its original shape after water absorption, which collagen is able to further maintain the distribution of hydroxyapatite on its surface and/or inside.
FIG. 4 shows an electron microscope photograph of the internal morphology of the SIS/HA composite bone repair material. It can be seen that hydroxyapatite is uniformly distributed on the surface and/or inside the collagen. The collagen scaffold can effectively maintain hydroxyapatite, realize the restoration after the implantation, and the collagen scaffold can be completely degraded. The bone filling material with porous structure and plastic characteristic is prepared by compounding hydroxyapatite particles and collagen from porcine small intestine submucosa. The method adopts the self-assembly characteristic of collagen, and uniformly wraps hydroxyapatite particles in the collagen fiber three-dimensional network, so that the material cannot be dispersed and broken in the using and transporting processes. The preparation process does not add any chemical cross-linking agent or adopt high-temperature cross-linking, retains the biological activity of collagen and the bone guiding property of hydroxyapatite, and the addition of the collagen enhances the biological activity of the bone filling material, and compared with the bone filling material which only takes the hydroxyapatite as the raw material, the repair effect is more obvious. In addition, the collagen can guide and promote the adhesion and proliferation of cells, accelerate the regeneration of blood vessels in the defect area and provide sufficient nutrient supply for the regeneration of bones in the defect area. Meanwhile, the self-assembled bone repair material has good hydrophilicity and can be well attached to a tissue defect area by the collagen. Finally, the hydroxyapatite particles used are stable in performance and are slowly absorbed by the human body, mainly through osseointegration, but do not cause changes in the pH of the surrounding environment, relative to the degradable calcium phosphate materials.
FIG. 5 shows a compression performance analysis of SIS/HA bone repair material with collagen to hydroxyapatite mass ratios of 1:29, 1:19, 1:14 and 1:9, respectively; the collagen accounts for 3.3 percent, 5 percent, 6.7 percent and 10 percent of the bone repair material by mass respectively. Fig. 5 shows that as the collagen content increases, the compressive capacity of the bone repair material increases first and then decreases, the compressive capacity of the bone repair material is the best when the mass ratio of the collagen to the hydroxyapatite is 1:14, and in addition, the bone repair material still has better compressive capacity and remains blocky under high pressure without collapse due to pressure when the bone repair material has other ratios. By adopting the self-assembly characteristic of collagen, hydroxyapatite particles are uniformly wrapped inside the collagen fiber three-dimensional network, so that the material cannot be dispersed and cracked in the using and transporting processes. The preparation process does not add any chemical cross-linking agent or adopt high-temperature cross-linking, retains the biological activity of collagen and the bone guiding property of hydroxyapatite, and the addition of the collagen enhances the biological activity of the bone filling material, and compared with the bone filling material which only takes the hydroxyapatite as the raw material, the repair effect is more obvious.
Fig. 6 shows the results of the leachate cytotoxicity assays (p <0.05, p <0.01, ns means no statistical significance), where SIS/HA represents the composite bone repair material, HA represents the hydroxyapatite starting material, and control represents the blank. As can be seen from the figure, compared with the blank group, both the composite bone repair material and the hydroxyapatite material can ensure that the fibroblasts have good activity and can effectively complete the repair after being implanted.
The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A preparation method of a bone repair material, wherein the preparation method comprises the following steps:
a dissolving step: preparing a solution comprising collagen at a first temperature;
a dispersing step: dispersing hydroxyapatite in the dissolved solution, and uniformly stirring at a first temperature to obtain a mixed solution;
an incubation step: placing the mixed solution in an isotropic pressure environment, and incubating at a second temperature to form a collagen scaffold; the hydroxyapatite particles are loaded in the collagen scaffold and/or on the surface of the collagen scaffold to complete the self-assembly of the hydroxyapatite and the collagen;
a freeze-drying step: carrying out freeze drying treatment on the product of the incubation step to obtain the bone repair material;
wherein the first temperature is 2-10 ℃, preferably 4 ℃; the second temperature is 20-40 deg.C, preferably 37 deg.C.
2. The preparation method according to claim 1, wherein the dissolving step comprises:
dissolving collagen in a pre-cooled phosphate buffer at a first temperature to obtain a collagen-containing solution; wherein the concentration of collagen in the dissolving solution is 0.5-2% (w/v), preferably 1% (w/v).
3. The process according to claim 1 or 2, wherein the collagen is derived from small intestine submucosa, preferably porcine small intestine submucosa.
4. The production method according to any one of claims 1 to 3, wherein the incubation step comprises:
and placing the mixed solution into a pre-cooled mold, applying isotropic pressure to the mold for briquetting, and incubating at a second temperature for 10-60min to complete the self-assembly of the hydroxyapatite and the collagen.
5. The preparation method according to any one of claims 1 to 4, wherein the lyophilizing step comprises: placing the bone repair material into a cold trap of a freeze dryer for precooling for 60-120min, starting a vacuum pump after precooling, and carrying out vacuum drying, wherein the temperature of the cold trap is set to-35 ℃ to-45 ℃, and is preferably-40 ℃.
6. A bone repair material, wherein the bone repair material comprises:
a collagen scaffold having a three-dimensional porous structure;
hydroxyapatite particles loaded inside, and/or on the surface of, the collagen scaffold; preferably, the hydroxyapatite particles are uniformly loaded on the surface and/or inside the collagen scaffold; preferably, the bone repair material is prepared by a method according to any one of claims 1 to 5.
7. The bone repair material according to claim 6, wherein the collagen scaffold has a mass fraction of 3.3-10% and the hydroxyapatite particles have a mass fraction of 90-96.7% based on the total mass of the bone repair material.
8. The bone repair material according to claim 6 or 7, wherein the porosity of the collagen scaffold is not less than 80%, and the particle size of the hydroxyapatite particles is 100-150 μm.
9. The bone repair material according to any one of claims 6 to 8, wherein the collagen scaffold is formed from collagen derived from the small intestine submucosa.
10. Use of a bone repair material according to any one of claims 6 to 9, or prepared according to the method of any one of claims 1 to 5, as or in the preparation of a bone filler material as one or more of: reserving alveolar sites after tooth extraction, filling alveolar bone defects, or filling bone defects at any non-bearing positions;
optionally, the bone defect at the non-weight bearing location is selected from one or more of: bone defects of maxillofacial surgery, bone defects of dental surgery, skull defects and various closed fracture bone defects of four limbs.
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