CN117695439B - Periosteum-like material and preparation method and application thereof - Google Patents
Periosteum-like material and preparation method and application thereof Download PDFInfo
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- CN117695439B CN117695439B CN202311447542.9A CN202311447542A CN117695439B CN 117695439 B CN117695439 B CN 117695439B CN 202311447542 A CN202311447542 A CN 202311447542A CN 117695439 B CN117695439 B CN 117695439B
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 134
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- Medicinal Preparation (AREA)
Abstract
The invention discloses a periosteum-like material and a preparation method and application thereof. The periosteum-like material is prepared by compounding natural polymer material sodium alginate with dopamine hydrochloride, bone organic component type I collagen and bone inorganic component nano hydroxyapatite and optimizing reaction conditions (solution concentration, reaction time, reaction temperature and the like). Compared with a pure sodium alginate film, the periosteum-like material designed by the invention has the advantages of reduced swelling performance and improved mechanical property; the periosteum has good surface rigidity, can provide good adhesion growth conditions for cells, has good cell compatibility for bone marrow mesenchymal stem cells and vein endothelial cells, has the capability of promoting blood vessel formation and bone marrow mesenchymal stem cell osteogenic differentiation, and can also be used as an excellent substrate for loading exosomes (participating in intercellular communication, regulating and controlling tissue regeneration and repair and a new generation of revolutionary drug carrier).
Description
Technical Field
The invention belongs to the technical field of functional materials, and particularly relates to a periosteal-like material, and a preparation method and application thereof.
Background
Bone defects caused by trauma, tumor resection or infection are common problems in clinic, and bone defects often accompany damage to periosteal tissue on the surface of bone. In natural bone tissue, periosteum coated on the surface of bone is important in the growth and development of bone and the maintenance of normal functions, and is used as an important interface to increase the fusion between bone and surrounding tissues, and is a key for bone tissue regeneration, especially bone integration, bone modeling and bone remodeling in the bone defect repair process. Therefore, research and development of the bionic material with periosteum-like structure and function are the basis for realizing the fusion of the material and the organism tissue and repairing bone defects. The ideal periosteum material has the characteristics of good tissue compatibility, angiogenesis promotion, cell proliferation and differentiation promotion, mechanical property similar to that of normal periosteum tissue, matching of the material degradation speed with the tissue regeneration speed and the like.
At present, researchers mostly adopt natural or synthetic polymer materials for constructing bionic periosteum, wherein the natural polymer materials mainly comprise chitosan, collagen, sodium alginate, gelatin and the like; the synthetic polymer material mainly comprises polyethylene glycol, polyethylene oxide, poly (vinyl alcohol), poly (acrylic acid), poly (2-hydroxyethyl methacrylate), polylactic acid, polyglycolic acid, polycaprolactone and the like. The periosteum-like material constructed by the natural polymer material has good biomechanical property but lower bioactivity; the natural polymer material has good biological activity, but has insufficient mechanical properties.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary aim of the invention is to provide a preparation method of periosteal-like materials. According to the preparation method, natural high polymer material sodium alginate is adopted to be compounded with dopamine hydrochloride, bone organic component type I collagen (Col I) and bone inorganic component nano hydroxyapatite (nHAP), and a composite membrane material with good adhesiveness and ductility is prepared by optimizing reaction conditions (solution concentration, reaction time and reaction temperature), and then the periosteal-like material with good mechanical property and biological activity is obtained by iron ion treatment.
The second aim of the invention is to provide the periosteal-like material prepared by the preparation method, and the periosteal-like material has good mechanical property, biocompatibility and bioactivity.
A third object of the present invention is to provide the use of the periosteal like material described above.
The primary purpose of the invention is realized by the following technical scheme:
A preparation method of periosteum-like material, which comprises the following steps,
(1) Preparation of an aqueous alginate gel: dissolving natural polymer Sodium Alginate (SA) in deionized water, and stirring at room temperature to prepare sodium alginate solution; adding nano hydroxyapatite (nHAP) into a sodium alginate solution, and performing ultrasonic dispersion; adding a carboxyl activating agent and a carboxyl stabilizing agent, uniformly stirring, adding dopamine hydrochloride (DA. HCl), continuously uniformly stirring, pouring into a mould, standing to form gel, and preparing alginic acid hydrogel;
(2) Preparation of alginic acid/collagen composite film: soaking the alginic acid hydrogel in the step (1) in bone organic component type I collagen (Col I), fully dialyzing, and drying to form a film to prepare an alginic acid/collagen composite film;
(3) Construction of periosteal-like materials: and (3) sequentially soaking the alginic acid/collagen composite membrane in the step (2) in a calcium chloride solution and an iron chloride solution to prepare the periosteum-like material.
Preferably, the carboxyl activating agent in step (1) is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (edc·hcl); the carboxyl stabilizer is N-hydroxysuccinimide (NHS), and the molar mass ratio of the carboxyl activator to the carboxyl stabilizer is 1:1.
Preferably, the molar mass ratio of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, the dopamine hydrochloride and the sodium alginate is 1:1:2 to 3; the molar mass ratio of the nano hydroxyapatite to the sodium alginate is 1:25 to 30.
Preferably, the mass concentration of the sodium alginate solution in the step (1) is 1.5% -2%, preferably 2%.
Preferably, in the step (1), the molar mass ratio of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, the dopamine hydrochloride and the sodium alginate is 1:1:2; the molar mass ratio of the nano hydroxyapatite to the sodium alginate is 1:25.
Preferably, in the step (1), the ultrasonic temperature is 20-25 ℃, and the ultrasonic time is 30-40 min.
Preferably, the ultrasonic temperature in the step (1) is 20 ℃, and the ultrasonic time is 30min.
Preferably, the concentration of the bone organic component type I collagen in step (2) is 1 to 1.5%, preferably 1%.
Preferably, the dialysis time in the step (2) is 24-48 hours, and the drying temperature is 45-50 ℃; the drying time is 48 hours, and the soaking time is 12-24 hours.
Preferably, the dialysis time in step (2) is 48 hours and the drying temperature is 45 ℃; the drying time is 48 hours, and the soaking time is 24 hours.
Preferably, in the step (3), the mass concentration of the calcium chloride solution is 1-2%, and the soaking time is 4-6 hours; the mass concentration of the ferric chloride solution is 0.1-0.2%, and the soaking time is 18-24 h.
Preferably, in the step (3), the mass concentration of the calcium chloride solution is 2%, and the soaking time is 6 hours; the mass concentration of the ferric chloride solution is 0.2%, and the soaking time is 24 hours.
The second object of the invention is achieved by the following technical scheme:
The periosteum-like material prepared by the preparation method has good mechanical property, biocompatibility and bioactivity.
The third object of the invention is achieved by the following technical scheme:
An application of periosteum-like material in bone defect repair process.
The invention adopts natural polymer material Sodium Alginate (SA) and dopamine hydrochloride (DA.HCl), bone organic component type I collagen and bone inorganic component nano hydroxyapatite (nHAP) to compound, prepares the composite membrane material with good adhesiveness and ductility by optimizing reaction conditions (solution concentration, reaction time, reaction temperature and the like), and then obtains the periosteal-like material (organic/inorganic hybrid membrane material) with good mechanical property and biological activity by iron ion treatment. Wherein, the film material is endowed with good formability and mechanical properties (flexibility) by constructing a double cross-linked network platform based on covalent bonds and ionic bonds; the membrane material is endowed with good adhesive property through the introduction of dopamine; the natural bone organic component Col I and the bone inorganic component nHAP are added into the membrane material to endow the membrane material with good biocompatibility; the high-valence iron ions are introduced into the membrane material to carry out ion exchange in molecular chains, so that on one hand, the mechanical strength (tensile strength) of the membrane material is effectively improved, the problems of increased membrane brittleness and sharply reduced mechanical strength caused by easy decrosslinking of the alginic acid membrane material in a culture medium are effectively solved, and on the other hand, the introduction of the iron ions with proper concentration can effectively promote angiogenesis and bone regeneration.
Compared with the prior art, the invention has the following advantages:
(1) The novel organic/inorganic periosteum-like material with good mechanical property, biocompatibility and blood vessel and bone regeneration induction capability is innovatively developed by taking various polymer materials as base materials, and compared with a pure sodium alginate film, the swelling property of the novel polymer periosteum-like material is reduced, and the mechanical property is improved;
(2) The periosteum-like membrane prepared by the invention has good surface rigidity, can provide good adhesion growth conditions for cells, and has good cell compatibility for bone marrow mesenchymal stem cells and vein endothelial cells;
(3) In the preparation process of the periosteum-like material, the chelating ability of the molecular chain of sodium alginate and ferric iron ions is stronger than that of calcium ions, so that the concentration of ferric iron ions in cells can be reduced by chelating the ferric iron ions of vein endothelial cells, and the related angiogenesis factors are up-regulated presumably;
(4) The periosteum-like membrane prepared by the invention has the capability of promoting bone marrow mesenchymal stem cells to osteogenic differentiate.
(5) The periosteum-like material prepared by the invention can also be used as an excellent substrate for loading exosomes (participating in intercellular communication, regulating and controlling tissue regeneration and repair and a new generation of revolutionary drug carrier).
Drawings
FIGS. 1 (A) and 1 (B) are schematic gel forming diagrams of hydrogels prepared from 1% SA and 3% SA, respectively; FIG. 1 (C) is a schematic illustration of a water gel after nHAP is dispersed by magnetic stirring;
FIG. 2 (A) is a graph of contact angle measurements for SA/ColI and nHAP/SA/ColI films; FIG. 2 (B) is a stress-strain plot of SA/ColI and nHAP/SA/ColI films;
FIGS. 3 (A) and 3 (B) are general appearance diagrams of CaCO 3 @SA and nHAP@SA hydrogels, respectively; FIGS. 3 (C) and 3 (D) are graphs showing the activity of BMSCs grown on CaCO 3 @ SA/ColI and nHAP @ SA/ColI membranes, respectively, by live-dead fluorescent staining;
FIG. 4 (A) is a graph of contact angle measurements of different periosteal materials; FIG. 4 (B) is a graph of the water content of different periosteal materials; FIG. 4 (C) is a graph of swelling ratios of different periosteal materials; FIG. 4 (D) is a graph showing degradation rates of different periosteal materials;
FIG. 5 (A) shows the microscopic morphology of the periosteal surface of each group observed by a scanning electron microscope; FIG. 5 (B) shows the elemental composition of the periosteal surface of each group analyzed by an energy spectrometer; FIG. 5 (C) shows the result of detecting the periosteal like crystal structure by X-ray diffraction (XRD);
FIG. 6 (A) is a stress-strain curve for each set of periosteal-like membranes; FIG. 6 (B) is a morphology of 0.2% ferric chloride treated periosteum-like curl and recovery; FIG. 6 (C) is a surface hardness test of different periosteum types;
FIGS. 7 (A) and 7 (B) are graphs showing the proliferation activity of BMSCs and HUVECS on periosteum of human umbilical vein respectively; FIGS. 7 (C) and 7 (D) are graphs showing the activity of BMSCs and HUVECS growth on periosteum of each group, respectively, by live-dead fluorescent staining;
FIG. 8 (A) is a tube formation of HUVECs after different periosteal treatments (4 h); FIG. 8 (B) is a quantitative analysis result of the tubular structure in FIG. 8 (A);
FIG. 9 shows the results of WB assay of the expression and quantitative analysis of angiopoiesis marker proteins after co-culture of HUVECs with different periosteum classes;
FIG. 10 is a graph showing the Q-PCR analysis of the bone formation marker gene expression levels of BMSCs after 7 days of culture on different periosteum types;
fig. 11 (a) and 11 (B) are respectively an inverted fluorescence microscope and a scanning electron microscope for observing the load, distribution and microstructure of exosomes on different periosteum types.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1
The preparation method of the periosteal-like material comprises the following steps:
(1) Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, the mixture is stirred for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (the molar mass ratio of EDC.HCl to NHS is 1:1) are sequentially added, and dopamine hydrochloride (DA.HCl) is added after the uniform stirring, wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min;
(2) Preparation of alginic acid/collagen composite film: soaking SA hydrogel in 1% Col I for 24h, fully dialyzing for 48h, and drying at 45deg.C to form a film to obtain alginic acid/collagen composite film;
(3) Construction of periosteal-like materials: and (3) soaking the dried alginic acid/collagen composite membrane in a 2% calcium chloride solution for 6 hours, then soaking in a 0.2% ferric chloride solution for 24 hours, and fully cleaning to obtain the periosteum-like material (0.2% Fe).
Comparative example 1
Preparation of an aqueous alginate gel: under the condition of room temperature, dissolving natural polymer Sodium Alginate (SA) in deionized water, stirring for 12 hours to prepare SA solution with the mass concentration of 1% and 3%, then sequentially adding a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC.HCl to NHS is 1:1), stirring uniformly, and then adding dopamine hydrochloride (DA.HCl), wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; compared with example 1, it was found that gel formation was not possible under 1% SA, but 3% SA gave too viscous a hydrogel due to higher solution viscosity, which was detrimental to uniform stirring, see FIGS. 1 (A) and 1 (B).
Comparative example 2
Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC.HCl to NHS is 1:1) are sequentially added, and dopamine hydrochloride (DA.HCl) is added after stirring is carried out uniformly, wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, nhap: the molar mass ratio of SA is 1:5,1:30 and 1:50, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min; when nHAP is compared with example 1: the molar mass ratio of SA is 1:5 and 1: glue was not applied at 50, and nHAP: the molar mass ratio of SA is 1:30, the gel can be formed smoothly, and the mechanical strength of the hydrogel is nHAP: the molar mass ratio of SA is 1:25, and therefore nHAP: the molar mass ratio of SA is 1:25 to 30. The contact angle of water of the film material is detected by a contact angle measuring instrument, and compared with a pure alginic acid film (SA/ColI), the contact angle of the film (nHAP/SA/ColI) after nHAP is added is obviously reduced, which shows that the addition of nHAP improves the hydrophilicity of the film to a certain extent, and the specific view is shown in fig. 2 (A); FIG. 2 (B) is a stress-strain plot of SA/Col I and nHAP/SA/Col I, and it can be seen that the addition of nHAP significantly improves the mechanical strength of the film.
Comparative example 3
Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC.HCl to NHS is 1:1) are sequentially added, and dopamine hydrochloride (DA.HCl) is added after stirring is carried out uniformly, wherein the molar mass ratio of EDC.HCl, NHS to SA is 1:1:2, molar mass ratio of nhap to SA is 1:25, DA. HCl: the molar mass ratio of SA is 1:1 and 1:3, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min; in comparison with example 1, it was found that the addition amount of DA. HCl did not affect the formation of the hydrogel, but DA. HCl: SA is 1:1 has a mechanical strength of 1:2, da·hcl: SA is 1: hydrogels of 3 with 1:2, the mechanical strength of the hydrogel is not greatly different. Thus, DA. HCl: the molar mass ratio of SA is 1:2 to 3.
Comparative example 4
Preparation of an aqueous alginate gel: under the condition of room temperature, dissolving natural polymer Sodium Alginate (SA) in deionized water, stirring for 12 hours to prepare SA solution with the mass concentration of 2%, adding calcium carbonate (CaCO 3) into the SA solution, fully and uniformly dispersing CaCO 3 by utilizing ultrasound, then sequentially adding a carboxyl activator of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer of N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC.HCl to NHS is 1:1), stirring uniformly, and then adding dopamine hydrochloride (DA.HCl), wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of CaCO 3 to SA is 1: 5-6, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min; compared to example 1, the hydrogel turned dark brown in a short time and had poor adhesion, see in particular fig. 3 (a) and fig. 3 (B). From this, caCO 3 is not suitable as a calcium source. Then, compared with example 1, the cells inoculated on the membrane material are observed under a microscope after being stained by a live-dead staining kit, the cells are adhered and spread on the periosteum-like membrane taking CaCO 3 as a calcium source, the cell morphology is in a round shape, and the cell morphology does not have good cell compatibility, particularly, the cell morphology is shown in fig. 3 (C) and 3 (D), so that the CaCO 3 is further proved to be unsuitable as the calcium source.
Comparative example 5
Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, then nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is stirred uniformly by a magnetic stirrer, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) are sequentially added (wherein the molar mass ratio of EDC.HCl to NHS is 1:1), dopamine hydrochloride (DA.HCl) is added after the uniform stirring, and the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the magnetic stirring temperature is 20 ℃, and the magnetic stirring time is 30min; compared with example 1, nHAP was found to be not sufficiently dispersed in the SA solution by means of magnetic stirring, but distributed in the form of white small lumps in the hydrogel, and the hydrogel became dark brown in a short time and had poor adhesion, see in particular fig. 1 (C).
Comparative example 6
Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC.HCl to NHS is 1:1) are sequentially added, and dopamine hydrochloride (DA.HCl) is added after stirring is carried out uniformly, wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 37 ℃ and the ultrasonic time is 30min; in comparison with example 1, the solution was found to be unable to gel. Therefore, the selection of the ultrasonic temperature has a great influence on the gelation process of the hydrogel, and the proper ultrasonic temperature is required to be selected, and the ultrasonic temperature is better at 20-25 ℃ through testing.
Comparative example 7
Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC to NHS is 1:1) are sequentially added, and dopamine hydrochloride (DA.HCl) is added after stirring is carried out uniformly, wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃, and the ultrasonic time is 20min and 40min; compared with example 1, nHAP cannot be uniformly dispersed in SA solution when ultrasound is performed for 20min, but is distributed in a white small block shape in hydrogel; and nHAP is uniformly distributed in the hydrogel between 40min and 30min of ultrasound, and the mechanical strength of the hydrogel is not greatly different. Therefore, the selection of the ultrasonic time has a great influence on the dispersion degree of nHAP, the proper ultrasonic time is required to be selected, and the ultrasonic time is better within 30-40 min after test.
Comparative example 8
(1) Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC.HCl to NHS is 1:1) are sequentially added, and dopamine hydrochloride (DA.HCl) is added after stirring is carried out uniformly, wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min;
(2) Preparation of alginic acid/collagen composite film: soaking SA hydrogel in 0.5% and 1.5% Col I for 24 hr; compared to example 1, it was found that 0.5% Col I, due to the lower concentration, was insufficient for adequate interaction with the hydrogel internal molecules, resulting in weaker mechanical strength of the hydrogel; the mechanical strength of the hydrogel after being soaked in 1.5% ColI is not greatly different from that of the hydrogel after being soaked in 1% ColI, so that the 1% ColI is selected to be a proper concentration by comprehensively considering factors such as material cost, mechanical strength of the hydrogel and the like.
Comparative example 9
(1) Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC.HCl to NHS is 1:1) are sequentially added, and dopamine hydrochloride (EDC.HCl) is added after stirring is carried out uniformly, wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min;
(2) Preparation of alginic acid/collagen composite film: soaking SA hydrogel in 1% Col I for 24 hr; after full dialysis for 48 hours, drying and film forming are carried out at 60 ℃ to prepare the alginic acid/collagen composite film; the film was severely shrunken after drying, becoming smaller and thicker than in example 1. Therefore, the selection of a proper drying temperature is important for preparing the alginic acid/collagen composite membrane, and the detection shows that the drying temperature is proper at 45-50 ℃.
Comparative example 10
(1) Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC.HCl to NHS is 1:1) are sequentially added, and dopamine hydrochloride (DA.HCl) is added after stirring is carried out uniformly, wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min;
(2) Preparation of alginic acid/collagen composite film: soaking SA hydrogel in 1% Col I for 24h, fully dialyzing for 48h, and drying at 45deg.C to form a film to obtain alginic acid/collagen composite film;
(3) Construction of periosteal-like materials:
immersing the dried alginic acid/collagen composite membrane in a 1% calcium chloride solution for 6 hours; the mechanical properties of the films after soaking in 1% calcium chloride are inferior compared to example 1. Therefore, the concentration of the immersed calcium chloride solution has a great influence on the mechanical properties of the membrane, and the concentration of 2% of calcium chloride is suitable according to experimental tests.
Comparative example 11
(1) Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS) (wherein the molar mass ratio of EDC.HCl to NHS is 1:1) are sequentially added, and dopamine hydrochloride (DA.HCl) is added after stirring is carried out uniformly, wherein the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min;
(2) Preparation of alginic acid/collagen composite film: soaking SA hydrogel in 1% Col I for 24h, fully dialyzing for 48h, and drying at 45deg.C to form a film to obtain alginic acid/collagen composite film;
(3) Construction of periosteal-like materials:
Immersing the dried alginic acid/collagen composite membrane in 2% calcium chloride solution for 4 hours; compared with example 1, the calcium chloride solution cannot sufficiently permeate into the membrane to crosslink with alginic acid after soaking for 4 hours, so that the mechanical properties of the membrane are poor. Therefore, the soaking time of the 2% calcium chloride solution has great influence on the mechanical properties of the periosteal-like material, and the soaking time of the 2% calcium chloride solution is properly selected to be 6 hours according to experimental tests.
Comparative example 12
(1) Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS.HCl) are sequentially added (wherein the molar mass ratio of EDC.HCl to NHS is 1:1), dopamine hydrochloride (DA.HCl) is added after stirring is carried out uniformly, and the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min;
(2) Preparation of alginic acid/collagen composite film: soaking SA hydrogel in 1% Col I for 24h, fully dialyzing for 48h, and drying at 45deg.C to form a film to obtain alginic acid/collagen composite film;
(3) Construction of periosteal-like materials:
Soaking the dried alginic acid/collagen composite membrane in a 2% calcium chloride solution for 6 hours, then soaking in a 0.2% ferric chloride solution for 12 hours and 18 hours respectively, and then fully cleaning to obtain a periosteum-like material; compared with example 1, the mechanical properties of the periosteum are poor after soaking 0.2% ferric chloride for 12 hours, and it is possible that iron ions cannot sufficiently penetrate into the periosteum to crosslink with alginic acid; the mechanical properties of the periosteum-like film soaked for 18 hours are not greatly different from those soaked for 12 hours. Therefore, the soaking time of the ferric chloride has an influence on the mechanical property of the prepared periosteum-like material, and the selection of 18-24 hours is preferable.
Comparative example 13
(1) Preparation of an aqueous alginate gel: under the condition of room temperature, natural polymer Sodium Alginate (SA) is dissolved in deionized water, stirring is carried out for 12 hours to prepare SA solution with the mass concentration of 2%, nano hydroxyapatite (nHAP) is added into the SA solution, nHAP is fully and uniformly dispersed by utilizing ultrasound, then a carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and a carboxyl stabilizer N-hydroxysuccinimide (NHS.HCl) are sequentially added (wherein the molar mass ratio of EDC.HCl to NHS is 1:1), dopamine hydrochloride (DA.HCl) is added after stirring is carried out uniformly, and the molar mass ratio of EDC. HCl, NHS, DA.HCl to SA is 1:1:1:2, molar mass ratio of nhap to SA is 1:25, continuously stirring uniformly, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel; the ultrasonic temperature is 20 ℃ and the ultrasonic time is 30min;
(2) Preparation of alginic acid/collagen composite film: soaking SA hydrogel in 1% Col I for 24h, fully dialyzing for 48h, and drying at 45deg.C to form a film to obtain alginic acid/collagen composite film;
(3) Construction of periosteal-like materials:
And (3) soaking the dried alginic acid/collagen composite membrane in a 2% calcium chloride solution for 6 hours, then soaking in 0%, 0.1%, 1% and 2% ferric chloride solutions for 24 hours, and fully cleaning to obtain the periosteal-like material (0% Fe, 0.1% Fe, 1% Fe and 2% Fe).
Test experimental example 1
The materials prepared in example 1 and comparative example 13 were tested.
(1) Contact angle test
In order to examine the hydrophilicity and hydrophobicity of each group of membrane materials, the water contact angle of the membrane materials was measured by a contact angle measuring instrument, as shown in fig. 4 (a), the contact angle of the alginic acid membrane after compositing with collagen was significantly reduced compared with that of the pure alginic acid membrane, indicating that the alginic acid membrane has better hydrophilicity, while the contact angle of the membrane surface after treatment with iron ions was increased, indicating that the hydrophilicity of the membrane was reduced to some extent by the iron ions treatment, probably due to the increase of the surface roughness of the membrane after the iron ions treatment. Overall, the membrane material still has good hydrophilicity after treatment with iron ions.
(2) Water content and swelling ratio
To examine the water content of each group of film materials, the wet and freeze-dried films were weighed and recorded as m 1 and m 2, respectively, and the water content of each group of films was calculated by using formula (1). As shown in fig. 4 (B), the water content of the film after the iron ion treatment was significantly reduced compared with the untreated film, especially when the iron ion concentration was not less than 0.2%, the water content of the film was significantly lower than that of the untreated group and the 0.1% iron ion treated group, but the water content of the film between the high concentration iron ion treated groups was not significantly different, which further proves that the iron ion treatment increased the crosslinking degree of the film.
To examine the swelling ratio of each group of film materials, the surface moisture of the wet film was dried and then weighed and recorded as m 0. Then, each group of films was immersed in PBS, placed in a 37 ℃ incubator, the films were taken out at a predetermined time point, the surface moisture was wiped off, weighed again, recorded as m t, and the swelling ratios of the different film materials were calculated using formula (2). As a result, as shown in FIG. 4 (C), the swelling ratio of the film after the iron ion treatment was significantly reduced compared with the untreated film, and particularly when the iron ion concentration was not less than 0.2%, the swelling ratio of the film was significantly lower than that of the untreated group and the 0.1% iron ion treated group, and the swelling ratio of the film was continuously reduced as the iron ion concentration was increased, and when the time reached 48 to 96 hours, the swelling ratio of each group of films was maximized.
(3) Degradability test
To examine the degradation of each group of membrane materials in physiological environment, the lyophilized membranes were weighed and recorded as m 0. Then, each group of membranes was immersed in sterile PBS, placed in an incubator at 37 ℃, membranes were washed once with pure water at intervals (3 d, 7d, 14d, 21 d), lyophilized and weighed, denoted m t, and the degradation rate of the different membrane materials in PBS was calculated using formula (3). As a result, as shown in fig. 4 (D), the degradation rate of the film material after the iron ion treatment was significantly reduced as compared with the untreated group before 21 days, and the degradation rate of the film was also reduced as the concentration of the iron ion was increased, but no significant difference was seen in the degradation rate of the film between the concentrations of 0.1% and 0.2% and between the concentrations of 1% and 2%. When the soaking time reaches 60 days, the degradation rate of each group of membranes reaches the highest, and no obvious difference is found among the membrane groups.
(4) Scanning electron microscope, energy spectrum analysis and X-ray diffraction pattern
The film surface structure and elemental composition were observed and analyzed by Scanning Electron Microscopy (SEM) and energy spectroscopy (EDS), respectively. As can be seen from fig. 5 (a), the distribution of the particles nHAP is seen on the surface of each group of film materials, but the particle size, density and coarseness of the film surface are significantly increased after the treatment of the iron ions, which is probably due to the fact that the substitution of the iron ions reduces the crystallinity of nHAP and simultaneously increases the crosslinking degree of the film. Further spectral analysis results also show that, specifically, as shown in fig. 5 (B), the calcium element in the film is partially replaced by iron element after the iron ion treatment, the energy peak gradually decreases with the increase of the iron ion concentration, and the energy peak of iron element gradually increases.
In order to detect the components and the crystal structure of each group of film materials, the film materials are subjected to freeze drying and then are subjected to scanning analysis by an X-ray diffractometer, as shown in fig. 5 (C), before ferric chloride is soaked, a sharp characteristic peak appears on the film at 37 degrees, the corresponding peak intensity after ferric chloride soaking treatment is obviously reduced, and the more obvious reduction along with the increase of the concentration of ferric ions, the film crystallinity is proved to be reduced after the ferric ions are treated, the internal cross-linked network is more compact, and the crystallinity is gradually reduced along with the increase of the concentration of ferric ions.
(5) Mechanical property test
After the surface moisture of each group of film materials is wiped, the tensile strength of the film in a wet state is detected by using a universal mechanical testing machine. As can be seen from fig. 6 (a), the iron ion treatment significantly improved the tensile strength of the film compared to the untreated group, and when the iron ion concentration was less than 0.2%, the tensile strength of the film increased with the increase in the iron ion concentration. However, when the iron ion concentration is higher than 0.2%, the film tensile strength decreases instead as the iron ion concentration increases. In addition, when the iron ion concentration is higher than 0.2%, the film brittleness is significantly increased, and when the iron ion concentration is lower than 0.2%, the film material still has good toughness (ductility), and it can be restored well to the pre-deformation state by twisting the film after the 0.2% ferric chloride treatment, as shown in fig. 6 (B). Further testing of the film hardness using a vickers hardness tester revealed that the film hardness after iron ion treatment was significantly increased, and the film surface hardness was also significantly increased with increasing iron ion concentration, but no significant difference was seen between the 0.1% and 0.2% iron ion treatment groups, as shown in fig. 6 (C).
(6) Cytotoxicity test
To examine the cell compatibility of each group of membrane materials, CCK-8 was used to examine the cell proliferation activity of venous endothelial cells (HUVECs) and bone marrow mesenchymal stem cells (BMSCs) on each group of membranes. HUVECs and BMSCs were inoculated onto each set of membranes, respectively, and incubated at 37℃under 5% CO 2 and 95% relative humidity, with liquid changes every three days. The membrane material was transferred to a new well plate at a predetermined time point, CCK-8 working solution (CCK-8 reagent: α -MEM basal medium=1:10) was added, and after incubation for 4 hours at 37 ℃ in an incubator, the light absorption value (OD) of the mixed solution at 450nm was detected by a microplate reader.
To examine the growth of cells on each group of membranes, the cells were inoculated on the membranes and cultured for 1 and 3 days, and then the cells on each group of membranes were subjected to live/dead fluorescent staining with a live/dead cell staining kit, and observed under a fluorescent inverted microscope. The BMSCs result is shown in the graph 7 (A) and the graph 7 (C), the HUVECs result is shown in the graph 7 (B) and the graph 7 (D), and by comparing the growth conditions of two cells on each group of membranes, the adhesion of the two cells on the membranes which are not treated by iron ions is poor, the proliferation rate is low, the cells can be well adhered and proliferated on the membranes which are treated by iron ions with different concentrations, and no obvious difference is found among the membranes of each group; from the fluorescence image results, it can be further seen that BMSCs and HUVECs have poor adhesion on untreated membranes, the cell morphology is mostly round, and cells can adhere, stretch and proliferate well on membranes treated by iron ions with different concentrations, and have normal fusiform morphology, and no obvious difference is found among the groups of membranes.
(7) Influence of periosteal like Material on vascularization
HUVECs were inoculated onto commercial matrigel at a density of 4X 10 4, incubated at 37℃under 5% CO 2 and 95% relative humidity for 2 hours, and then each group of membranes was added to the well plate, wherein the group without membrane was set as a blank control, and after continuing to incubate for 2 hours, the HUVECs cells were observed under an inverted microscope for tube formation, photographed, recorded and quantitatively analyzed, and the results are shown in FIG. 8 (A) and FIG. 8 (B). Compared with a blank control group, the membrane material treated by different iron ions has good angiogenesis promoting effect, and particularly, when the iron ion concentration is less than or equal to 0.2%, the tube promoting effect is enhanced along with the increase of the iron ion concentration, but when the iron ion concentration is more than 0.2%, the tube promoting effect of the membrane is reduced along with the increase of the iron ion concentration.
Furthermore, the expression of the vascular marker proteins CD31, HIF-1. Alpha. And VEGF in HUVES on each membrane group was further examined by WB technique, and quantitative analysis was performed, and the results are shown in FIG. 9. Compared with a blank control group, the vascular protein expression level of HUVES on the film treated with different iron ion concentrations is basically consistent with the result of a tube forming experiment, when the iron ion concentration is less than or equal to 0.2%, the vascular protein expression level of cells on the film is enhanced along with the increase of the iron ion concentration, but when the iron ion concentration is more than 0.2%, the vascular protein expression level is reduced along with the increase of the iron ion concentration.
(8) Influence of periosteal-like Material on osteogenesis
To examine the osteogenic differentiation promoting ability of each group of membrane materials, BMSCs cells were inoculated on each group of membranes at a density of 1×10 5, cultured overnight at 37 ℃, 5% CO 2 and 95% relative humidity, and osteogenic induction culture was performed the next day, once every three days, intracellular RNA was extracted after 7 days and the expression of intracellular osteogenic marker genes such as ALP, OCN, OPN and RUNX 2 was detected by RT-qPCR technique, and as a result, each group of membranes had an effect of promoting osteogenic differentiation of BMSCs cells, when the iron ion concentration was less than or equal to 0.2%, the intracellular osteogenic gene expression level was increased with the increase of the iron ion concentration, but when the iron ion concentration was >0.2%, the intracellular osteogenic gene expression level was decreased with the increase of the iron ion concentration, and the trend was substantially consistent with the membrane angiogenesis promoting trend.
(9) Testing of periosteal like material loaded exosomes
In order to visually observe the loading and distribution conditions of exosomes on the surfaces of all groups of membranes, after the exosomes are marked by using fluorescent dye PKH26, the exosomes are respectively incubated with 0% Fe and 0.2% Fe periosteum, after 24 hours, the exosomes which are not loaded on the membranes are washed off by PBS, and the exosomes loaded by the 0.2% Fe periosteum are obviously more and uniformly dispersed on the membranes, and only sporadically dispersed exosomes are distributed on the 0% Fe membrane as seen in an inverted fluorescence microscope; further, the microscopic morphology of the exosome loaded on the periosteum of 0.2% was observed by a Scanning Electron Microscope (SEM), and as shown in fig. 11 (B), compared with the dense block structure of the surface of the exosome-loaded film, a large amount of uniform granular exosome distribution on the film was seen after the exosome was loaded, and the exosome still maintained its good microscopic morphology.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (6)
1. The preparation method of the periosteum-like material is characterized by comprising the following steps of,
(1) Preparation of an aqueous alginate gel: dissolving natural polymer sodium alginate in deionized water, and stirring at room temperature to prepare sodium alginate solution; adding nano hydroxyapatite into sodium alginate solution, and performing ultrasonic dispersion; adding a carboxyl activating agent and a carboxyl stabilizing agent, uniformly stirring, adding dopamine hydrochloride, continuously uniformly stirring, pouring into a mould, standing to form gel, and preparing the alginic acid hydrogel;
(2) Preparation of alginic acid/collagen composite film: soaking the alginic acid hydrogel in the step (1) in bone organic component type I collagen, fully dialyzing, and drying to form a film to prepare an alginic acid/collagen composite film;
(3) Construction of periosteal-like materials: sequentially soaking the alginic acid/collagen composite membrane in the step (2) in a calcium chloride solution and an iron chloride solution to prepare a periosteum-like material;
The carboxyl activating agent in the step (1) is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, the carboxyl stabilizing agent is N-hydroxysuccinimide, and the molar mass ratio of the carboxyl activating agent to the carboxyl stabilizing agent is 1:1, a step of;
the molar mass ratio of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, the dopamine hydrochloride and the sodium alginate is 1:1:2 to 3; the molar mass ratio of the nano hydroxyapatite to the sodium alginate is 1: 25-30 parts;
the concentration of the bone organic component type I collagen in the step (2) is 1 to 1.5 percent;
The mass concentration of the calcium chloride solution in the step (3) is 1-2%; the mass concentration of the ferric chloride solution is 0.1-0.2%;
the mass concentration of the sodium alginate solution in the step (1) is 1.5-2%.
2. The method for preparing periosteal like material according to claim 1, wherein the ultrasonic temperature in the step (1) is 20-25 ℃ and the ultrasonic time is 30-40 min.
3. The method for preparing a periosteal like material according to claim 1, wherein the dialysis time in the step (2) is 24 to 48 hours, and the drying temperature is 45 to 50 ℃; the drying time is 48 hours, and the soaking time is 12-24 hours.
4. The method for preparing periosteal like material according to claim 1, wherein the soaking time of the calcium chloride solution in the step (3) is 4-6 hours; the soaking time of the ferric chloride solution is 18-24 hours.
5. A periosteal like material prepared by the preparation method according to any one of claims 1 to 4.
6. Use of the periosteal like material of claim 5 in a bone defect repair procedure.
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