CN107929812B

CN107929812B - Preparation method of biomimetic mineralized collagen scaffold

Info

Publication number: CN107929812B
Application number: CN201711195275.5A
Authority: CN
Inventors: 张旭; 林明丽; 沈敏娟; 刘欢欢; 王浩荣; 李彦秋
Original assignee: STOMATOLOGICAL HOSPITAL TIANJIN MEDICAL UNIVERSITY
Current assignee: Bonager Technology (Tianjin) Co.,Ltd.
Priority date: 2017-11-24
Filing date: 2017-11-24
Publication date: 2020-12-25
Anticipated expiration: 2037-11-24
Also published as: CN107929812A

Abstract

The invention provides a preparation method of a biomimetic mineralized collagen scaffold, which comprises the following steps of 1) preparation of biomimetic mineralized liquid: adjusting the pH of the carboxymethyl chitosan-amorphous calcium phosphate (or amorphous strontium phosphate or amorphous strontium carbonate) nanocomposite solution to be lower than the isoelectric point of CMC; 2) preparing a biomimetic mineralized collagen scaffold: mixing the bionic mineralization solution and the acid-soluble type I collagen, putting the mixture into a dialysis bag, sealing the bag, putting the bag into a PBS (phosphate buffer solution), self-assembling and mineralizing the bag for 2 to 5 days, adding an alkaline CMC/ACP (ASP or ASC) solution into the dialysis bag, and replacing the PBS buffer solution; and dialyzing the dialysis bag in deionized water for 10-72 h after 1-3 days, replacing the deionized water for many times, centrifuging the solution in the bag to obtain collagen gel, and freeze-drying to obtain the biomimetic mineralized collagen scaffold. The invention adopts the synergy of collagen self-assembly and mineralization, realizes the mineralization in collagen fibers with higher efficiency, can integrate strontium element with osteogenesis promoting capability into a collagen scaffold, and has wide clinical application prospect.

Description

Preparation method of biomimetic mineralized collagen scaffold

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method of a biomimetic mineralized collagen scaffold.

Background

With the improvement of economic level and the maturity of implant restoration technology, the implant restoration has the advantages of no harm to adjacent teeth, beautiful appearance, comfort and the like, so that the implant restoration is widely applied to oral clinic. The bone condition of the implanted area has important significance on the osseointegration of the implant, so the treatment of the extraction socket after the tooth extraction is performed due to the tooth body and periodontal diseases is very important. In order to prevent and reduce the absorption of alveolar bone, a bone tissue repair material row site preservation operation can be placed in the tooth extraction pit, and the bone tissue repair material plays a good role in occupying and supporting in the alveolar ridge site preservation operation; in addition, oral and maxillofacial cysts and invalid cavities left after tumor resection are often the main reasons for delayed healing of wounds, and the bone tissue repair material is placed into the invalid cavities to eliminate the invalid cavities so as to promote bone union to obtain good effect; periodontal disease results in the resorption of alveolar bone also requiring the filling of bone tissue repair material to slow and stop the progression of the disease. And thus the demand for bone tissue repair materials is increasing. The autogenous bone in the bone tissue repair material has limited source and secondary damage to the autogenous bone, and allogeneic bone possibly has immunological rejection, thereby attracting a plurality of researchers to put the eyes on the bionic mineralization material to repair the bone tissue defect. The mineralized collagen scaffold can be used as a bone regeneration promoting material and has great potential and significance for prevention, diagnosis and treatment of oral diseases.

Natural bone tissue is a complex composed of an ordered arrangement of about 28% type I collagen fibers and 67% hydroxyapatite. The collagen fiber is formed by self-assembling collagen microfibrils, collagen molecules are staggered 1/4 to form an array to form the collagen microfibrils, the void region of the collagen molecules is rich in non-collagen, the collagen fibers are modified by the non-collagen, a mineralized precursor is stabilized, amorphous calcium phosphate is deposited in the collagen and converted into intra-fiber hydroxyapatite, and hydroxyapatite crystals are arranged along the c axis of the collagen fibers and then grow gradually. Mineralized collagen fibers are basic tissue units of bones, have a complex hierarchical structure and a precise assembly mode, and the highly ordered structure of the mineralized collagen fibers enables bone tissues to have unique hardness, toughness, strength and fracture resistance. These superior properties motivate us to study biomaterials that mimic bone-like or new functional materials.

The natural bone tissue also contains some trace elements, mainly strontium, which has bidirectional regulation effect and not only has the capability of promoting bone synthesis, but also has the capability of preventing bone mass loss. The action mechanism is that the activity of alkaline phosphatase can be increased, the synthesis of collagen and non-collagen in osteoblasts is increased, and the formation of bone is promoted by stimulating osteoblasts. Meanwhile, the bone resorption can be obviously influenced, the activity of the bone resorption can be inhibited by directly acting with osteoclast, and the loss of bone mass can be reduced. Strontium can also regulate the internal structure of hydroxyapatite, improve the hardness of hydroxyapatite, widen the size of bone crystals and increase the strength of bones. The strontium-doped hydroxyapatite can promote the adhesion and proliferation of osteoblasts, and has good biocompatibility and no obvious cytotoxicity.

The non-collagen plays an important role in the biomineralization process, plays an important regulation role in stabilizing precursors and inducing the nucleation and hierarchical self-assembly of mineral salts in the collagen scaffold, but has limited supply and high price. It is therefore essential to find biomimetic analogues with superior properties to mimic the non-collagenous effects. The carboxymethyl chitosan is a water-soluble chitosan derivative, is similar to non-collagen, is an amphoteric polyelectrolyte, has amphoteric ionization balance, good biocompatibility and biodegradability, is low in price, and has wide application prospects in the fields of medicines, foods, cosmetics and the like.

The traditional method for mineralizing collagen firstly limits a mineral substance nucleation and growth framework through a collagen self-assembly process, calcium and phosphorus particles smaller than 40nm enter collagen through the actions of static electricity, capillary action and the like to form mineralized collagen fibers, and a novel preparation method is developed in view of the characteristics of amphoteric polyelectrolyte of non-collagen analogue carboxymethyl chitosan, so that the mineralizing step is simplified, and the mineralizing efficiency is improved.

Disclosure of Invention

In view of the above, the invention aims to provide a preparation method of a biomimetic mineralized collagen scaffold, so as to overcome the defects of the prior art, the invention adopts the cooperative operation of collagen self-assembly and mineralization, not only simplifies the mineralization preparation process, but also breaks through the limitation that the particle size of amorphous calcium phosphate is less than 40nm, and small-particle-size ACP is aggregated into large-particle-size ACP (>40nm) under an acidic condition and can be deposited in collagen to form mineralized collagen.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a preparation method of a biomimetic mineralized collagen scaffold comprises the following steps,

1) preparing a biomimetic mineralized liquid: adjusting the pH value of the CMC/ACP solution of the carboxymethyl chitosan-amorphous calcium phosphate nano-composite to be lower than the isoelectric point of CMC by acid; preferably, the acid solution is hydrochloric acid or nitric acid;

2) preparing a biomimetic mineralized collagen scaffold: mixing the acidic carboxymethyl chitosan-amorphous calcium phosphate nano-composite solution obtained in the step 1) with acid-soluble type I collagen, putting the mixture into a dialysis bag, sealing the bag, putting the bag into PBS buffer solution, self-assembling and mineralizing the solution for 2 to 5 days, adding an alkaline CMC/ACP solution into the dialysis bag, replacing the PBS buffer solution outside the dialysis bag, treating the solution for 1 to 3 days, putting the dialysis bag into deionized water, dialyzing the solution for 10 to 72 hours, replacing the deionized water for multiple times, centrifuging the solution in the dialysis bag to obtain collagen gel, and finally freeze-drying the gel to obtain the bionic mineralized collagen scaffold.

The carboxymethyl chitosan used in the invention is a non-collagen bionic analogue with excellent performance, and CMC is similar to non-collagen, is an amphoteric polyelectrolyte, and can stabilize a mineralized precursor under an acidic condition by utilizing the characteristics of the carboxymethyl chitosan to guide collagen mineralization.

Preferably, in the step 1), the CMC-amorphous calcium phosphate nano-composite (CMC/ACP) solution contains 2-5 mg/ml of CMC and K₂HPO₄1mM to 6mM of CaCl₂·2H₂The concentration of O is 2 mM-10 mM; Ca/P molar ratio 1: 1-2: 1; and the pH was adjusted below 3 with HCl. The pH of the CMC/ACP solution in the step 1)And (3) slowly dripping HCl until the pH value of the mixed system is lower than 3, wherein the HCl is 6-8. The particle size of the ACP nano-particles is about 10-100 nm as observed by a transmission electron microscope.

When the pH value of the CMC/ACP solution is adjusted by the HCl, precipitates are separated out from the solution along with the reduction of the pH value, the most precipitates are obtained in an isoelectric point region (3-5), and then the precipitates are gradually dissolved. The CMC/ACP solution was clear at pH < 3.

Preferably, the preparation method of the carboxymethyl chitosan-amorphous calcium phosphate nano-composite solution comprises the steps of adding carboxymethyl chitosan into deionized water at normal temperature, stirring until the carboxymethyl chitosan is completely dissolved, and adding K₂HPO₄Obtaining solution A, adding CaCl₂·2H₂Dropwise adding an O aqueous solution into the A solution to form a carboxymethyl chitosan-amorphous calcium phosphate nano-composite (CMC/ACP) solution; in the step 1), the carboxymethyl chitosan is one or more than two of N-carboxymethyl chitosan, O-carboxymethyl chitosan and N, O-carboxymethyl chitosan, the viscosity of an aqueous solution of the carboxymethyl chitosan is 30-200, and the isoelectric point is about 3-5.

Preferably, in step 2), the acid-soluble type I collagen and the CMC/ACP solution are mixed in a volume ratio of 1: 1-1: 5, mixing; dissolving type I collagen with acid, wherein the acid comprises one or more of 0.1-0.5M hydrochloric acid, nitric acid or acetic acid; preferably, the type I collagen is type I rat tail collagen, cow skin collagen, pig skin collagen and fish skin collagen.

Preferably, the particle size of the amorphous calcium phosphate in the acidic carboxymethyl chitosan-amorphous calcium phosphate nano composite solution obtained in the step 1) is 10-100 nm.

Preferably, in the step 2), the cut-off molecular weight of the dialysis bag is 1000-14000; the buffer range of the PBS buffer solution is 7.2-7.4; with the balance of ions inside and outside the dialysis bag, the pH value in the dialysis bag gradually rises, collagen molecules gradually self-assemble and are combined with ACP, and ACP wrapped in collagen fibers gradually converts to HAP and grows to form mineralized collagen with a strict grade structure.

After self-assembly and mineralization are carried out for 2-4 days, the amount of the added alkaline CMC/ACP solution is 1/2-2 times of the volume of the solution in the dialysis bag; freezing the collagen gel at-20 to-40 ℃ for 24 to 48 hours, and freeze-drying to obtain the biomimetic mineralized collagen scaffold; preferably, the pH of the alkaline CMC/ACP solution is 8-12.

Preferably, in step 1), amorphous strontium phosphate or amorphous strontium carbonate is used instead of amorphous calcium phosphate.

Preferably, the preparation of the carboxymethyl chitosan-amorphous strontium phosphate nano-composite solution comprises the following steps of adding carboxymethyl chitosan into deionized water at normal temperature, stirring until the carboxymethyl chitosan is completely dissolved, and adding K₂HPO₄To obtain solution A, adding SrCl₂·6H₂Dropwise adding the O aqueous solution into the A solution to form a carboxymethyl chitosan-amorphous strontium phosphate nano composite (CMC/ASP) solution;

preferably, the carboxymethyl chitosan is one or more than two of N-carboxymethyl chitosan, O-carboxymethyl chitosan and N, O-carboxymethyl chitosan.

Preferably, the carboxymethyl chitosan-amorphous strontium phosphate nano-composite (CMC/ASP) solution contains 2-5 mg/ml of CMC and K₂HPO₄1mM to 10mM, SrCl₂·6H₂The concentration of O is 5 mM-20 mM; the molar ratio of Sr to P is 1: 1-2: 1; and adjusting the pH to less than 3 with HCl;

the particle size of amorphous strontium phosphate in the acidic carboxymethyl chitosan-amorphous strontium phosphate nano composite solution obtained in the step 1) is 5-50 nm.

Preferably, the preparation of the carboxymethyl chitosan-amorphous strontium carbonate nanocomposite solution comprises the following steps of adding carboxymethyl chitosan into deionized water at normal temperature, stirring until the carboxymethyl chitosan is completely dissolved, and adding Na₂CO₃To obtain solution A, adding SrCl₂·6H₂Dropwise adding an O aqueous solution into the A solution to form a carboxymethyl chitosan-amorphous strontium carbonate nanocomposite (CMC/ASC) solution;

Preferably, the CMC-amorphous strontium carbonate nanocomposite (CMC/ASC) solution contains 2-5 mg/ml of CMC,K₂HPO₄1mM to 10mM, SrCl₂·6H₂The concentration of O is 5 mM-20 mM; the molar ratio of Sr to P is 1: 1-2: 1; and adjusting the pH to less than 3 with HCl;

the grain diameter of the amorphous strontium carbonate in the acidic carboxymethyl chitosan-amorphous strontium carbonate nano composite solution obtained in the step 1) is 5-50 nm.

The invention also provides application of the biomimetic mineralized collagen scaffold prepared by the method in bone defect repair materials.

The natural bone tissue has a typical highly complex hierarchical structure, and nowadays, many scholars are dedicated to research and simulation of the natural bone tissue to research a novel biomimetic mineralized collagen material with superior performance to solve the current situation of short supply and short demand of bone graft materials. The organic matrix of bone is type I collagen, hydroxyapatite is deposited in collagen to form a complex, non-collagen plays a crucial role in regulating collagen mineralization, the non-collagen has an isoelectric point, and carboxymethyl chitosan (CMC) is amphoteric polyelectrolyte, so that the net charge of a carboxymethyl chitosan solution is positive when the net charge is lower than the isoelectric point, and the net charge of the carboxymethyl chitosan solution passes through-NH when the net charge is lower than the isoelectric point₃ ⁺…PO₄ ^3-Coordination also stabilizes the mineralized precursor, mineralizing the collagen. And the CMC has good biocompatibility, degradability and no toxic or side effect, and guides the inside and outside of the fiber to be mineralized cooperatively. The acid mineralized liquid and the collagen in a molecular state are combined under an acid condition, the pH is increased to promote the conversion of the mineralized precursor to a crystal state, and meanwhile, the pH is required to be increased by collagen self-assembly, so that the pH is gradually increased by adopting the mineralized liquid-collagen mixed liquid stabilized by carboxymethyl chitosan under the environment with low pH, and the mineralized collagen fibers are formed by self-assembly and mineralization at the same time.

Compared with the prior art, the preparation method of the biomimetic mineralized collagen scaffold has the following advantages:

(1) the biomimetic mineralized collagen scaffold prepared by the method is a mineralized material with a highly complex hierarchical structure, the pore region and the overlapping region are periodically alternated mutually, hydroxyapatite is orderly combined in the pore region of the collagen fiber and grows along the long axis of the collagen fiber, and a bone graft material similar to the components and the structure of natural bone tissues is constructed. The hydroxyapatite is deposited in the collagen to nucleate and grow while the collagen molecules are self-assembled, and mineralized collagen fibers with orderly deposited crystals are formed.

(2) The method of the invention can prepare the cross striation structure (D is 67nm) which is characterized by natural collagen fiber, and hydroxyapatite crystals are regularly deposited in the collagen pore region for nucleation and growth.

(3) The invention adopts carboxymethyl chitosan (CMC) as a calcium-phosphorus stabilizer, is more similar to non-collagen, is an amphoteric polyelectrolyte, has positive net charge when the pH of the solution is lower than the isoelectric point of CMC, can attract phosphate radical ions through electrostatic action so as to stabilize ACP, has negative net charge when the pH of the solution is higher than the isoelectric point of CMC, and can attract calcium ions through electrostatic action so as to stabilize ACP, so that the pH is adjusted to be lower than the isoelectric point of CMC, and the ACP can also be stabilized. And the CMC has excellent performances of biocompatibility, naturalness, no toxic or side effect and the like, is easy to obtain and has low price.

(4) According to the invention, the ACP stabilized by the acidic carboxymethyl chitosan is adopted, the particle size of the ACP is 10-100 nm, and the limitation of collagen mineralization on the particle size of the ACP of less than 40nm is broken through due to the synergistic effect of collagen self-assembly and mineralization. ACP in the dialysis bag under an acidic condition is directly contacted with collagen in a molecular state, the precondition that collagen self-assembly and mineralization are carried out simultaneously is constructed, the dialysis bag is placed in PBS buffer solution, positive ions and negative ions inside and outside the dialysis bag gradually tend to be balanced, an environment which gradually rises from low pH is created for the collagen, collagen molecules are self-assembled into collagen fibers along with the rise of the pH, and amorphous calcium phosphate deposited in the collagen is gradually converted into hydroxyapatite.

(5) The mineralized liquid adopted by the invention can also be SrCl₂·6H₂O instead of CaCl₂·2H₂O, or simultaneously with Na₂CO₃In place of K₂HPO₄The principle of which is in connection with CaCl₂·2H₂And the same O, and the superior performance of the collagen scaffold is utilized to prepare the collagen scaffold with higher hardness and strength.

Drawings

FIG. 1 is a graph of the turbidity at various pH values of CMC solutions prepared in example 1 of the present invention.

Wherein the numbers in the figure represent the pH values.

FIG. 2 is a Transmission Electron Microscope (TEM) observation of CMC/ACP nanoparticles prepared in example 1 of the present invention;

FIG. 3 is a Transmission Electron Microscope (TEM) observation of collagen fibers prepared in example 1 of the present invention with a mineralization time of 1 hour;

FIG. 4 is a Transmission Electron Microscope (TEM) observation of collagen fibers prepared in example 1 of the present invention with a mineralization time of 3 days;

FIG. 5 is a Transmission Electron Microscope (TEM) observation of collagen fibers prepared in example 1 of the present invention with a mineralization time of 5 days;

FIG. 6 shows the collagen fibers prepared in example 1 of the present invention (mineralized for 3 days) observed by a field emission Scanning Electron Microscope (SEM).

FIG. 7 is a photograph of a collagen scaffold prepared in example 1 of the present invention.

FIG. 8 shows the CMC/ASP nanoparticles prepared in example 2 of the present invention, observed by Transmission Electron Microscopy (TEM).

FIG. 9 shows the collagen fibers prepared in example 2 of the present invention (mineralized for 3 days) observed by Transmission Electron Microscope (TEM).

FIG. 10 shows the collagen fibers prepared in example 3 of the present invention (mineralized for 3 days) observed by Transmission Electron Microscope (TEM).

Fig. 11 is a Transmission Electron Microscope (TEM) observation of the collagen fibers prepared in comparative example 1.

Fig. 12 shows the collagen fibers prepared in comparative example 2, observed by Transmission Electron Microscopy (TEM).

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to examples.

Example 1

(1) At all times200mg of O-carboxymethyl chitosan (viscosity: 45, Mw: 13700, isoelectric point 3.6) was added to 30ml of deionized water with sufficient stirring by a warm magnetic stirrer, and after complete dissolution, 41.76mg of K was added₂HPO₄Obtaining solution A; 58.8mg CaCl were added to 10ml deionized water₂·2H₂O, forming a liquid B; dropwise adding the solution B into the solution A under the conditions of normal temperature and full stirring of a magnetic stirrer to form a carboxymethyl chitosan-amorphous calcium phosphate nano-composite (CMC/ACP) solution; the CMC/ACP solution was adjusted to pH 2 with HCl. The transmission electron microscope shows that the characteristic structure is shown in figure 2, and the size of the amorphous calcium phosphate nano-particles is about 40-100 nm. SAED is shown without concentric circular feature rings.

(2) Soaking a dialysis bag in 50% alcohol by volume fraction for 30 minutes, washing with deionized water, sealing one end, taking 1ml of I-type rat tail collagen, putting the I-type rat tail collagen into the dialysis bag, taking 3ml of the acidic CMC/ACP solution, putting the acidic CMC/ACP solution into a dialysis bag with the molecular weight cutoff of 8000-14000, and uniformly mixing. After sealing, the cells were placed in 15ml of PBS buffer, mineralized for 1 hour, at which time the pH was about 2.6, and characterized by transmission electron microscopy as shown in FIG. 3, the collagen self-assembly process was observed, at which time hydroxyapatite was deposited along the C-axis. Mineralization is carried out for 3 days, the pH value is about 6, and the transmission electron microscope characterization is shown in figure 4, so that collagen self-assembly is completed, and hydroxyapatite is orderly deposited in a pore region. After 3 days, a CMC/ACP solution was prepared again, the pH thereof was adjusted to 11 with NaOH, 3ml of the CMC/ACP solution was placed in a dialysis bag, and the PBS buffer outside the dialysis bag was replaced. And observed after 2 days. The characterization results are shown in fig. 5 and 6, and fig. 5 shows that hydroxyapatite crystals grow along the long axis of the collagen fibers. SAED can see crystalline diffraction rings. As shown in figure 6, the diameter of the collagen fiber is about 600nm, the dialysis bag is placed in deionized water for dialysis for 10-72 h, the solution in the dialysis bag is centrifuged after the deionized water is replaced for multiple times to obtain collagen gel, and finally the gel is frozen and dried to obtain the biomimetic mineralized collagen scaffold. The collagen scaffold is shown in figure 7.

Example 2

200mg of O-carboxymethyl chitosan (viscosity: 45, Mw: 13700, isoelectric point 3.6) was added to 30ml of deionized water under sufficient stirring with a magnetic stirrer at room temperature, and 62.7mg of K was added after complete dissolution₂HPO₄Obtaining solution A; in 10ml160.2mgSrCl is added into the ionized water₂·6H₂O, forming a liquid B; under the conditions of normal temperature and full stirring of a magnetic stirrer, dropwise adding the solution B into the solution A to form a carboxymethyl chitosan-amorphous strontium phosphate nano composite (CMC/ASP) solution; the CMC/ASP solution pH was adjusted to 2 with HCl. The transmission electron microscope is characterized in that the structure is shown in figure 8, and the size of the CMC/ASP nano-particles is about 5-50 nm.

Soaking the dialysis bag in 50% alcohol for 30 minutes, washing with deionized water, sealing one end, taking 1ml of I-type rat tail collagen, putting the I-type rat tail collagen into the dialysis bag, taking 3ml of the acidic CMC/ASP liquid, putting the acidic CMC/ASP liquid into the dialysis bag with the cut-off molecular weight of 8000-14000, and uniformly mixing. After sealing, 15ml of PBS buffer solution is added for mineralization for 3 days, and the characterization results are shown in FIG. 9.

Example 3

200mg of O-carboxymethyl chitosan (viscosity: 45, Mw: 13700, isoelectric point 3.6) was added to 30ml of deionized water under sufficient stirring with a magnetic stirrer at room temperature, and after complete dissolution, 25.44mg of Na was added₂CO₃Obtaining solution A; 106mg SrCl was added to 10ml deionized water₂·6H₂O, forming a liquid B; under the conditions of normal temperature and full stirring of a magnetic stirrer, dropwise adding the solution B into the solution A to form a carboxymethyl chitosan-amorphous strontium carbonate nano composite (CMC/ASC) solution; the CMC/ASC solution was adjusted to pH 2 with HCl.

Soaking the dialysis bag in 50% alcohol for 30 minutes, washing with deionized water, sealing one end, taking 1ml of I-type rat tail collagen, putting the I-type rat tail collagen into the dialysis bag, taking 3ml of the acidic CMC/ASP solution, putting the acidic CMC/ASP solution into the dialysis bag with the cut-off molecular weight of 8000-14000, and uniformly mixing. After sealing, 15ml of PBS buffer solution is added for mineralization for 3 days, and the characterization results are shown in FIG. 10.

Comparative example 1

This comparative example provides collagen self-assembled and then mineralized in a neutral mineralized fluid.

Placing acid-soluble type I rat tail collagen in PBS buffer solution, changing PBS buffer solution once a day, dipping self-assembled collagen with copper mesh after 3 days, drying, and floating in neutral CMC/ACP mineralized liquid for 3 days. The characterization result is shown in fig. 11, and the hydroxyapatite crystals in the fiber are deposited and the amorphous calcium phosphate outside the fiber is aggregated.

Comparative example 2

This comparative example provides a neutral CMC/ACP solution mineralized collagen.

In substantially the same manner as in example 1 except that the pH was not adjusted after the preparation of the biomimetic mineralization solution (CMC/ACP solution) of step (2) of example 1, the pH was adjusted to 7 after mixing it with the acid-soluble type i rat tail collagen. The characterization result is shown in figure 12, and the periodic striations can be seen, and no obvious hydroxyapatite crystal deposition exists.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A preparation method of a biomimetic mineralized collagen scaffold is characterized by comprising the following steps: comprises the following steps of (a) carrying out,

1) preparing a biomimetic mineralized liquid: adjusting the pH value of the CMC/ACP solution of the carboxymethyl chitosan-amorphous calcium phosphate nano-composite to be lower than the isoelectric point of CMC by acid; the acid solution is hydrochloric acid or nitric acid;

2) preparing a biomimetic mineralized collagen scaffold: mixing the acidic carboxymethyl chitosan-amorphous calcium phosphate nano-composite solution obtained in the step 1) with acid-soluble type I collagen, putting the mixture into a dialysis bag, sealing the bag, putting the bag into PBS buffer solution, and self-assembling and mineralizing the collagen in the bag for 2-5 days; then adding an alkaline CMC/ACP solution into the dialysis bag, replacing a PBS buffer solution outside the dialysis bag, dialyzing the dialysis bag in deionized water for 10-72 hours after treating for 1-3 days, replacing the deionized water for multiple times, centrifuging the solution in the dialysis bag to obtain collagen gel, and finally freeze-drying the collagen gel to obtain the biomimetic mineralized collagen scaffold;

in the step 1), the CMC/ACP solution of the carboxymethyl chitosan-amorphous calcium phosphate nano-composite CMC/ACP contains 2-5 mg/ml of CMC and K₂HPO₄1mM to 6mM of CaCl₂·2H₂The concentration of O is 2 mM-10 mM; Ca/P molar ratio 1.666: 1; and the pH was adjusted below 3 with HCl.

2. The method for preparing a biomimetic mineralized collagen scaffold according to claim 1, wherein: the preparation method of the carboxymethyl chitosan-amorphous calcium phosphate nano-composite solution comprises the steps of adding carboxymethyl chitosan into deionized water at normal temperature, stirring until the carboxymethyl chitosan is completely dissolved, and adding K₂HPO₄Obtaining solution A, adding CaCl₂·2H₂Dropwise adding the O aqueous solution into the A solution to form a CMC/ACP solution of the carboxymethyl chitosan-amorphous calcium phosphate nano-composite; the carboxymethyl chitosan in the step 1) is one or more than two of N-carboxymethyl chitosan, O-carboxymethyl chitosan and N, O-carboxymethyl chitosan.

3. The method for preparing a biomimetic mineralized collagen scaffold according to claim 1 or 2, wherein: in the step 2), the acid-soluble type I collagen and the CMC/ACP solution are mixed according to the volume ratio of 1: 1-1: 5, mixing; dissolving type I collagen with acid, wherein the acid comprises one or more of 0.1-0.5M hydrochloric acid, nitric acid or acetic acid; the type I collagen is rat tail collagen, cow skin collagen, pig skin collagen and fish skin collagen;

the particle size of amorphous calcium phosphate in the acidic carboxymethyl chitosan-amorphous calcium phosphate nano composite solution obtained in the step 1) is 10-100 nm.

4. The method for preparing a biomimetic mineralized collagen scaffold according to claim 1 or 2, wherein: in the step 2), the cut-off molecular weight of the dialysis bag is 1000-14000; the buffer range of the PBS buffer solution is 7.2-7.4; after self-assembly and mineralization are carried out for 2-4 days, the amount of the added alkaline CMC/ACP solution is 0.5-2 times of the volume of the solution in the dialysis bag; freezing the collagen gel at-20 to-40 ℃ for 24 to 48 hours, and freeze-drying to obtain a biomimetic mineralized collagen scaffold; the pH value of the alkaline CMC/ACP solution is 8-12.

5. The method for preparing a biomimetic mineralized collagen scaffold according to claim 1, wherein: in the step 1), a carboxymethyl chitosan-amorphous strontium phosphate nano-composite CMC/ACP solution or a carboxymethyl chitosan-amorphous strontium phosphate nano-composite CMC/ACP solution is used for replacing the carboxymethyl chitosan-amorphous calcium phosphate nano-composite CMC/ACP solution.

6. The method for preparing a biomimetic mineralized collagen scaffold according to claim 5, wherein: the preparation method of the carboxymethyl chitosan-amorphous strontium phosphate nano compound solution comprises the following steps of adding carboxymethyl chitosan into deionized water at normal temperature, stirring until the carboxymethyl chitosan is completely dissolved, and adding K₂HPO₄To obtain solution A, adding SrCl₂·6H₂Dropwise adding the O aqueous solution into the A solution to form a CMC/ASP solution of the carboxymethyl chitosan-amorphous strontium phosphate nano-composite;

the carboxymethyl chitosan is one or more of N-carboxymethyl chitosan, O-carboxymethyl chitosan and N, O-carboxymethyl chitosan.

7. The method for preparing a biomimetic mineralized collagen scaffold according to claim 6, wherein: the CMC/ASP solution of the carboxymethyl chitosan-amorphous strontium phosphate nano compound contains 2-5 mg/ml of CMC and K₂HPO₄1mM to 10mM, SrCl₂·6H₂The concentration of O is 5 mM-20 mM; the molar ratio of Sr to P is 1.666: 1; and adjusting the pH to less than 3 with HCl;

the particle size of amorphous strontium phosphate in the acidic carboxymethyl chitosan-amorphous strontium phosphate mixed solution obtained in the step 1) is 5-50 nm.

8. The method for preparing a biomimetic mineralized collagen scaffold according to claim 5, wherein: the preparation method of the carboxymethyl chitosan-amorphous strontium carbonate nano-composite solution comprises the steps of adding carboxymethyl chitosan into deionized water at normal temperature, stirring until the carboxymethyl chitosan is completely dissolved, and adding Na₂CO₃To obtain solution A, adding SrCl₂·6H₂Dropwise adding the O aqueous solution into the A solution to form a CMC/ASC solution of the carboxymethyl chitosan-amorphous strontium carbonate nano composite;

9. The method for preparing a biomimetic mineralized collagen scaffold according to claim 8, wherein: the CMC/ASC solution of carboxymethyl chitosan-amorphous strontium carbonate nano composite contains 2-5 mg/ml of CMC and Na₂CO₃1mM to 10mM, SrCl₂·6H₂The concentration of O is 5 mM-20 mM; the molar ratio of Sr to P is 1.666: 1; and adjusting the pH to less than 3 with HCl;

10. The application of the biomimetic mineralized collagen scaffold prepared by the method according to any one of claims 1 to 9 in bone defect repair materials.