CN112546305A - Ordered porous composite material and preparation method and application thereof - Google Patents
Ordered porous composite material and preparation method and application thereof Download PDFInfo
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- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- B33—ADDITIVE MANUFACTURING TECHNOLOGY
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2430/00—Materials or treatment for tissue regeneration
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
Abstract
The invention provides an ordered porous composite material, a preparation method and application thereof. The composite artificial bone has photosensitive property, can be quickly crosslinked and cured under ultraviolet irradiation, has good mechanical property, controllable degradation speed and high bone induction activity, and can be used as a composite artificial bone integrating bone and alveolar bone repair and treatment; and can effectively induce vascularization and rapidly induce the growth and formation of new bones in situ, thereby achieving better effect on the aspect of promoting bone regeneration. The preparation method of the ordered porous composite material provided by the invention is simple and easy to operate, can be formed at room temperature and is convenient to apply.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to an ordered porous composite material and a preparation method and application thereof.
Background
Both chronic periodontitis and aging are prone to progressive loss of bone mass. Progressive bone loss can lead to tooth movement and dislocation, and eventually even to tooth loss, thereby reducing the quality of life of the patient. Therefore, in situ regeneration of alveolar bone has attracted attention from scholars.
The natural bone matrix is composed of type I collagen and nano-hydroxyapatite. Based on the organic-inorganic composite characteristics of the natural bone matrix, currently, bone powder implantation is generally adopted clinically to guide tissue regeneration and realize in-situ regeneration of alveolar bone. Among them, autologous bone grafting is the gold standard, and although good results can be obtained, secondary trauma occurs and donor sites are limited, so that patient acceptance is low.
The new generation alveolar bone repair material takes a bionic bone matrix structure as a design concept and aims to promote the self-repair of bone tissues. However, the existing artificially synthesized polymer material has the disadvantages of overlong degradation period and lower biological activity. However, natural polymer materials suffer from problems of too fast degradation, low mechanical strength, potential immunogenicity and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a novel ordered porous composite material, a preparation method and application thereof by modifying the fibroin.
It is a first object of the present invention to provide an ordered porous composite scaffold to alleviate at least one of the technical problems of the prior art.
The second purpose of the invention is to provide the preparation method of the ordered porous composite scaffold, which is simple and easy to operate, can be formed at room temperature, has low forming temperature and is convenient to apply.
The third purpose of the invention is to provide the application of the ordered porous composite scaffold in bone tissue engineering and dental tissue engineering restoration.
The method is realized by the following technical scheme:
an ordered porous composite comprising photosensitive silk fibroin and bioactive glass dispersed in the photosensitive silk fibroin.
Further, the photosensitive silk fibroin is obtained by carrying out photopolymerization chemical crosslinking reaction modification on silk fibroin and glycidyl methacrylate.
Further, the weight ratio of the bioactive glass to the ordered porous composite material is 0.1-10%. If the content is less than 0.1%, the biological function expression of the bioactive glass is not obvious; if it is more than 10%, photocuring is not possible.
Further, the weight ratio of the bioactive glass to the ordered porous composite material is 1-8%, and preferably 3% -6%.
Further, the bioactive glass is borosilicate bioactive glass.
Further, the borosilicate bioactive glass is selected from one or more of the following compositions: aXO bB2O3·cP2O5·dSiO2；
Wherein, a, b, c and d are mole fractions, a is 20-66, b is 2-48, c is 2-10 and d is 30-76; x is Ca and/or Mg and/or Sr.
Preferably, the borosilicate bioactive glass comprises one or at least two of the following compositions:
10SrO·10CaO·10MgO·40B2O3·2P2O5·28SiO2、
20CaO·8MgO·27B2O3·4P2O5·41SiO2、
14SrO·20CaO·36B2O3·2P2O5·28SiO2、
14SrO·8CaO·8MgO·40B2O3·2P2O5·28SiO2、
8SrO·12CaO·8MgO·10B2O3·4P2O5·58SiO2、
14SrO·8CaO·8MgO·38B2O3·2P2O5·30SiO2、
10SrO·10CaO·10MgO·38B2O3·2P2O5·30SiO2、
20CaO·8MgO·27B2O3·4P2O5·41SiO2、
14SrO·18CaO·36B2O3·2P2O5·30SiO2、
20CaO·8MgO·30B2O3·4P2O5·38SiO2、
20CaO·20B2O3·2P2O5·58SiO2。
further, the particle size range of the bioactive glass is 30-300 nm. If the particle size of the bioactive glass is less than 30nm, the particles are too fine, and nano-size toxicity is possibly generated; if the particle size of the bioactive glass is larger than 300nm, the particles are too large, and the rheological property and the printability of the whole printing paste are influenced.
Preferably, the borosilicate bioactive glass also comprises ZnO and Ag2O, CuO and CeO2One or at least two of them. The above substances can provide other biological functions to bioactive glass, such as zinc ion can be combined with various coenzymes, silver ion can be antibacterial, copper ion can promote cardiovascular maintenance normal form and function, and cerium ion can promote cartilage regeneration.
Preferably, the ZnO content is 0-2 mol%, Ag 20 to 0.5 percent of O, 0 to 0.5 percent of CuO and CeO2The molar content of (A) is 0-1%.
Further, the pore size of the ordered porous composite material is 50-300 μm, preferably 70-150 μm; the ordered porous composite has a pore density of 50 to 300ppi, preferably 80 to 280ppi, more preferably 100-260 ppi. Appropriate pore size and pore density facilitate tissue ingrowth.
A preparation method of an ordered porous composite material comprises the following steps: mixing the photosensitive silk fibroin solution with bioactive glass to obtain slurry; and carrying out photocuring molding on the obtained slurry to obtain the ordered porous composite material.
Further, the weight ratio of the bioactive glass to the ordered porous composite material is 0.1-10%.
Preferably, the bioactive glass accounts for 1-8% of the weight of the ordered porous composite material, and more preferably 3-6%.
Further, the bioactive glass is borosilicate bioactive glass.
Further, in order to uniformly disperse the borosilicate bioactive glass in the photosensitive silk fibroin solution, the borosilicate bioactive glass can be firstly dispersed in 100-800 μ L of deionized water;
preferably, the borosilicate bioglass may be first dispersed in 500 μ L of deionized water.
Further, the specific method of the photocuring molding is as follows: and (3) placing the slurry into a photocuring three-dimensional forming printer, printing according to a preset model and parameters, and simultaneously carrying out photocuring curing.
Preferably, the preset model is a 3D model designed through a computer, is imported into slicing software for processing, and is stored as an stl format file;
further, the parameters are specifically: the slice thickness is 0.01-0.2mm, preferably 0.05 mm; the exposure time is 6-40s, preferably 30 s; the bottom exposure time is 30-80s, preferably 60 s; the number of the bottom layers is 2-6, preferably 3; the lifting distance of the Z axis is 4-8mm, preferably 6 mm; the Z-axis lifting speed is 2-7mm/s, preferably 3 mm/s. If the slice thickness is less than 0.01mm, the required printing time is long; if the thickness of the slice is more than 0.2mm, the printing precision is reduced, and the printed product may have obvious step feeling; the number of the bottom layers is related to the stability of the product in the initial stage, and if the number of the bottom layers is less than 2, the mechanical strength of the product in the initial stage is too weak; if the number of the bottom layers is more than 6, the initial stability of the product cannot be obviously enhanced; the lifting speed affects the peeling of the slurry from the surface of the product and the precision of the product.
Further, the time for the crosslinking is 10 to 300 seconds, preferably 30 to 60 seconds. If the crosslinking time is less than 10 seconds, the crosslinking is incomplete; if the crosslinking time is more than 300 seconds, no additional promotion effect is generated on the crosslinking process, so that the crosslinking time can be controlled between 10 and 300 seconds in order to save time.
In another implementation manner, the specific method of the photocuring molding is as follows: and (3) placing the slurry in a mold, and carrying out photo-crosslinking curing.
Further, the photosensitive silk fibroin solution is an aqueous solution of photosensitive silk fibroin.
Further, the photosensitive silk fibroin solution contains a photoinitiator.
Further, the content of the photoinitiator is 0.1-1%, preferably 0.2-0.5% of the photosensitive fibroin solution; less than 0.1% does not cause the article to cure, and the overall strength of the article increases with the addition of photoinitiator, but at the same time, the toxicity of the photoinitiator increases, so the photoinitiator content is preferably not more than 1%.
Preferably, the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate.
Further, the borosilicate bioactive glass is prepared by at least the following steps:
s1: dissolving hexadecyl ammonium bromide in deionized water, adding tetraethoxysilane and ammonia water, and stirring to obtain a mixed solution;
s2, adding tributyl borate and calcium nitrate tetrahydrate into the mixed solution obtained in the step S1, and reacting to obtain coarse micro-nano borosilicate bioactive glass;
and S3, washing, drying, preserving heat and cooling the coarse micro-nano borosilicate bioactive glass obtained in the step S2 to obtain the nano borosilicate bioactive glass.
Further, step S1 is performed in a water bath at a temperature of 30 to 50 ℃.
Further, before the reaction in step S2, one or more of silver nitrate, copper sulfate, or cerium nitrate may be added to the mixed solution.
Preferably, the reaction time of step S2 is 4-10 h.
The invention also provides the application of the ordered porous composite material in bone tissue engineering and dental tissue engineering.
The invention also provides a product of the ordered porous composite material prepared by the preparation method of the ordered porous composite material.
Compared with the traditional bone tissue repair material, dental tissue repair material and preparation method thereof, the invention has the following advantages:
1. the modified photosensitive silk fibroin-borosilicate bioactive glass composite material is combined with a photocuring three-dimensional forming printing technology, so that the natural bone matrix can be simulated more accurately.
2. The toughness and cell affinity of the material are improved through the photosensitive silk fibroin, the degradation speed of the material is regulated and controlled through borosilicate bioactive glass, the strength and osteoinductive activity of the material are improved, and the prepared ordered porous composite scaffold has good biodegradability, biocompatibility and osteoinductive activity.
3. Due to the introduction of borosilicate bioactive glass, compared with the traditional silk fibroin sponge dressing, the boron (B) precipitated from the ordered porous composite scaffold provided by the invention can regulate and control the bone loss caused by immunosuppression periodontitis, and a local slightly alkaline environment is formed at the same time, so that the invasion of acidophilic bacteria can be effectively resisted, and the scaffold has a certain antibacterial property.
4. The preparation method provided by the invention is simple and easy to operate, can be formed at room temperature and is convenient to apply.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1(a) shows the size and morphology of a micro-nano borosilicate bioactive glass according to the experimental analysis of the invention; FIG. 1(b) is an X-ray diffraction diagram of micro-nano borosilicate bioactive glass which is experimentally analyzed by the invention;
FIG. 2 is a graph illustrating the experimental analysis of the appearance of the ordered porous composite scaffold after freeze-drying;
FIG. 3(a) is a macroscopic view of an ordered porous composite scaffold provided by the experimental analysis of the present invention; FIG. 3(b) is a micro-topography of an ordered porous composite scaffold provided in example 6 of the present invention; fig. 3(c) is a graph illustrating the distribution of boron in the micro-nano borosilicate bioactive glass in the ordered porous composite scaffold according to an experimental analysis of the present invention; fig. 3(d) is a graph showing the distribution of the silicon element ordered porous composite scaffold in the micro-nano borosilicate bioactive glass according to the experimental analysis of the present invention;
FIG. 4 shows the weight loss results of two ordered composite porous scaffolds analyzed by the present invention, wherein the absorbance values of 8 per group (1d, 3d, 5d, 7d) are arranged in the order of Blank control, Sf, 1BG/Sli-MA, 2BG/Sli-MA, 3BG/Sli-MA, 4BG/Sli-MA, 5BG/Sli-MA and 6 BG/Sli-MA;
FIG. 5 shows the result of the cytotoxicity test of the three ordered porous composite scaffolds analyzed by the experiment of the present invention;
FIGS. 6(a) and (b) are graphs for analyzing the adhesion morphology of four periodontal ligament stem cells (PDLSCs) on the surface of an ordered porous composite scaffold according to the present invention; FIGS. 6(c) and (d) are fluorescence photographs of four periodontal ligament stem cells (PDLSCs) on the surface of the ordered porous composite scaffold according to the experimental analysis of the present invention;
FIG. 7 shows the result of the present invention's experiment analyzing the alkaline phosphatase (ALP) staining results of the five ordered porous composite scaffolds promoting the differentiation of periodontal ligament stem cells (PDLSCs).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The silk fibroin is a natural high molecular polymer and has the characteristics of good biocompatibility, biodegradability, no obvious antigenicity and the like. In addition, silk fibroin also HAs a certain mineralization capability, and can effectively induce Hydroxyapatite (HA) which is an inorganic mineral component of bones to mineralize and deposit on the surface, so that silk fibroin is gradually a popular material for bone tissue engineering repair in recent years.
The 3D printing technique utilizes three-dimensional data of Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) of an object by means of Computer Aided Design (CAD), precisely deposits materials through computer control, rapidly generates three-dimensional objects of arbitrary shapes, has significant advantages in the aspects of repeatability and flexibility of designing scaffolds, and precise control of three-dimensional scaffold structures (such as internal microstructures and macroscopic geometric structures), and the like, and has been widely applied in the construction of tissue engineering scaffolds. Among them, stereolithography Apparatus (SLA) has the characteristics of high precision and rapid prototyping.
However, due to the low viscosity of silk fibroin, the preparation of ink suitable for 3D printing cannot be realized, and further, the ordered porous scaffold cannot be effectively constructed for tissue engineering repair. Silk fibroin must therefore be chemically modified to improve its printability. The silk fibroin with photosensitive characteristic is obtained by utilizing photopolymerization chemical crosslinking reaction based on Glycidyl Methacrylate (GMA) modification technology, and the 3D printing performance of the silk fibroin can be effectively improved.
Although silk fibroin has certain mineralization property and tissue induction activity, silk fibroin is degraded too fast in vivo and is insufficient in mechanical strength. Therefore, an inorganic bioactive reinforcing phase needs to be introduced into the silk fibroin matrix, so that the degradation speed of the silk fibroin matrix is regulated and the silk fibroin matrix is endowed with proper mechanical strength. The bioactive glass is an inorganic bioactive material with excellent bone repair performance. After the bioactive glass is used for repairing bone tissues, the bioactive glass can be degraded in vivo and release bone metabolism nutrient ions of a matrix to form an ion and alkaline microenvironment. The local ion microenvironment can regulate and control the bone immune behavior, inhibit macrophage differentiation to osteoclast, mediate the directional osteogenesis and angioblast differentiation of stem cells, and finally obtain excellent bone repair performance. Meanwhile, through the design of components, particle size and micro morphology, the degradation speed, the ion precipitation speed and the formed local pH value of the bioactive glass can be regulated and controlled.
Although the bioactive glass has high osteoinductive activity, the bioactive glass has high brittleness and low elastic modulus, so that an inorganic-organic composite material needs to be constructed on the basis of bioactive glass particles, and further processed into a proper application form.
Therefore, the invention combines the respective advantages of the bioactive glass and the photosensitive silk fibroin, can more accurately simulate the natural bone matrix based on the photocuring stereolithography printing technology, improves the toughness and the cell affinity of the material through the photosensitive silk fibroin, and improves the strength and the osteoinductive activity of the material through the bioactive glass.
The ordered porous composite material takes silk fibroin as a matrix, and bioactive glass is dispersed in the silk fibroin matrix, wherein the bioactive glass can be dispersed on the surface of the silk fibroin matrix, can be dispersed in the silk fibroin matrix, such as porous pores, and can be dispersed on the surface of the silk fibroin matrix and in the porous pores simultaneously.
In the present invention, the ordered porous composite material comprises photosensitive silk fibroin, bioactive glass, optionally other components, and unavoidable impurities. Wherein the sum of the weight of the photosensitive silk fibroin, the bioactive glass, optionally other components and impurities is 100%. Preferably, the adopted bioactive glass is micro-nano borosilicate bioactive glass with a tiny size.
Wherein optionally the other components may be, for example, but not limited to, antibiotics, growth factors, small RNA molecules, hyaluronic acid, collagen or vitamins, among others, which contribute to tissue repair.
According to the invention, borosilicate bioactive glass is preferably used, and nano-grade micro-nano borosilicate bioactive glass is preferably used. The borosilicate bioactive glass has excellent bioactivity, biodegradability and biocompatibility, and can degrade and release important bone regeneration elements such as strontium (Sr), calcium (Ca), boron (B), magnesium (Mg), silicon (Si) and the like in human body fluid; after the degradation process is finished, the borosilicate bioactive glass can be converted into a calcium-phosphorus compound, can effectively adsorb protein and cells, and is favorable for stimulating mesenchymal stem cells to differentiate into osteoblasts.
Example 1
The embodiment provides an ordered porous composite material, which comprises photosensitive silk fibroin and borosilicate bioactive glass dispersed in the photosensitive silk fibroin. The borosilicate bioactive glass is 1% by weight, based on 100% by weight of the ordered porous composite scaffold.
Wherein the borosilicate bioactive glass has the following composition:
14SrO·8CaO·8MgO·38B2O3·2P2O5·30SiO2。
the particle size range of the borosilicate bioactive glass is 100-300 nm.
The borosilicate bioactive glass also comprises ZnO, and the molar content of the ZnO is 1%.
The pore size of the ordered porous composite material is 50-300 μm; the pore density was 50-300 ppi.
Example 2
The embodiment provides an ordered porous composite material, which is different from the embodiment 1 in that the weight of the micro-nano borosilicate bioactive glass is 3% based on 100% of the weight of the ordered porous composite scaffold.
The micro-nano borosilicate bioactive glass comprises the following components:
10SrO·10CaO·10MgO·38B2O3·2P2O5·30SiO2，
the particle size range of the bioactive glass is 30-100 nm.
The borosilicate bioactive glass also comprises Ag2O，Ag2The molar content of O is 0.25%.
The pore size of the ordered porous composite material is 200-300 mu m; the pore density was 200-300 ppi.
Example 3
The embodiment provides an ordered porous composite material, which is different from the embodiment 1 in that the weight of the micro-nano borosilicate bioactive glass is 5% based on 100% of the weight of the ordered porous composite scaffold.
The micro-nano borosilicate bioactive glass comprises the following components:
20CaO·8MgO·27B2O3·4P2O5·41SiO2，
the particle size range of the bioactive glass is 50-250 nm.
The borosilicate bioactive glass also comprises Ag2O、CuO，Ag2The molar content of O is 0.2%, and the molar content of CuO is 0.3%.
The pore size of the ordered porous composite material is 100-200 mu m; the pore density was 100-200 ppi.
Example 4
The embodiment provides an ordered porous composite material, which is different from the embodiment 1 in that the weight of the micro-nano borosilicate bioactive glass is 7% based on 100% of the weight of the ordered porous composite scaffold.
The micro-nano borosilicate bioactive glass comprises the following components:
14SrO·18CaO·36B2O3·2P2O5·30SiO2，
the particle size range of the bioactive glass is 150-250 nm.
The borosilicate bioactive glass also comprises Ag2O、CuO、CeO2，Ag2The molar content of O is 0.1%, the molar content of CuO is 0.4%, and CeO2In a molar amount of0.5％。
The pore size of the ordered porous composite material is 80-120 mu m; the pore density was 80-120 ppi.
Example 5
The embodiment provides an ordered porous composite scaffold, which is different from the embodiment 1 in that the weight of the micro-nano borosilicate bioactive glass is 0.1% based on 100% of the weight of the ordered porous composite scaffold.
The micro-nano borosilicate bioactive glass comprises the following components:
20CaO·8MgO·30B2O3·4P2O5·38SiO2，
the particle size range of the bioactive glass is 200-300 nm.
The pore size of the ordered porous composite material is 180-260 mu m; the pore density was 180-260 ppi.
Example 6
The embodiment provides an ordered porous composite scaffold, which is different from the embodiment 1 in that the weight of the micro-nano borosilicate bioactive glass is 10% based on 100% of the weight of the ordered porous composite scaffold.
The micro-nano borosilicate bioactive glass comprises the following components:
20CaO·20B2O3·2P2O5·58SiO2，
the particle size range of the bioactive glass is 80-150 nm.
The pore size of the ordered porous composite material is 100-150 mu m; the pore density was 100-150 ppi.
Experimental analysis of structure and morphology of ordered porous composite scaffold and micro-nano borosilicate bioactive glass thereof
In order to save cost, the borosilicate bioactive glass and the ordered porous composite material used in example 2 were selected for the experiment.
(1) The borosilicate bioactive glass powder used in example 2 was examined for microscopic appearance and infrared spectroscopy. The micro-topography and the infrared spectrum are respectively shown in FIG. 1(a) and FIG. 1 (b). As shown in FIG. 1(a), the borosilicate bioactive glass has a more regular spherical particle shape, and the particle size range is about 100-150 nm. Infrared results fig. 1(b) shows that borosilicate bioactive glass exhibits typical silicon-oxygen and boron-oxygen oscillation peaks.
(2) The ordered porous composite scaffolds made from the ordered porous composite material of example 2 were packaged in a packaging bag and sealed, and then sterilized by irradiation to obtain sterile ordered porous composite scaffolds, as shown in fig. 2.
(3) The ordered porous composite scaffold prepared from the ordered porous composite material of example 2 was subjected to freeze drying, then surface gold spraying, and macroscopic (see fig. 3(a)) and microscopic morphology (see fig. 3(b)) were observed by using an environmental scanning electron microscope. The results show that the prepared ordered porous composite scaffold has a porous structure; the microstructure showed an irregular honeycomb structure; the borosilicate bioactive glass is dispersed in the matrix of the ordered porous composite scaffold, and it can also be seen from fig. 3(c) and 3(d) that the borosilicate bioactive glass is distributed uniformly in the scaffold.
Example 7 preparation of ordered porous composite
(1) Preparation of borosilicate bioactive glass:
weighing borosilicate bioactive glass 8 SrO.12CaO.8MgO.10B according to molar ratio2O3·4P2O5·58SiO2The raw materials of the components. The preparation process comprises the following steps: the temperature of the water bath kettle is firstly adjusted to 40 ℃. Cetyl ammonium bromide was dissolved in 100ml of deionized water, stirred for 5 minutes, added with ethyl orthosilicate, and stirred for 5 minutes. After adding 3ml of 25% aqueous ammonia, the mixture was stirred for 10 minutes. And adding tributyl borate and calcium nitrate tetrahydrate for reaction for 4 hours. The product deionized water and absolute ethyl alcohol are alternately washed for 3 times. After drying, putting the powder into a muffle furnace, heating to 450 ℃ at the speed of 1 ℃ per minute, preserving the heat for 4 hours, and then cooling along with the furnace. Thus obtaining the micro-nano borosilicate bioactive glass.
(2) Preparation of photosensitive silk fibroin solution:
dissolving photosensitive silk fibroin in water, and uniformly mixing to obtain a 30% photosensitive silk fibroin solution; 0.1% of a photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate was added.
(3) Preparing an ordered porous composite scaffold:
in order to make borosilicate bioactive glass uniformly disperse in photosensitive silk fibroin solution, firstly, dispersing borosilicate bioactive glass in 500 mu L of deionized water; and mixing the prepared borosilicate bioactive glass with the silk fibroin solution to prepare silk fibroin/bioglass slurry with the borosilicate bioactive glass content of 1%, 2%, 3%, 4%, 5% and 6%, and printing by 3D to obtain the ordered porous composite scaffold which is named as 1BG/Sli-MA, 2BG/Sli-MA, 3BG/Sli-MA, 4BG/Sli-MA, 5BG/Sli-MA and 6BG/Sli-MA respectively.
The 3D printing comprises the following specific steps:
placing the slurry into a stereolithography Apparatus (SLA) printer, designing a 3D model through a computer, introducing into slicing software, processing, and storing as a stl (stereolithography) format file, wherein the thickness of the slice is set to be 0.01 mm; the exposure time was 6 s; the bottom exposure time was 30 s; the number of the bottom layers is 2; the lifting distance of the Z axis is 4 mm; the Z-axis lifting speed was 2 mm/s.
And printing the ordered porous composite material according to a pre-designed model and corresponding parameters.
(4) And placing the prepared ordered porous composite material in a photocuring instrument for photocuring and crosslinking for 1min to obtain an ordered porous composite scaffold product.
The prepared ordered porous composite material can be applied to bone tissue engineering and dental tissue engineering. Specifically, the ordered porous composite material can be used for preparing a bracket and a composite artificial bone.
Example 8
(1) Preparation of borosilicate bioactive glass:
weighing borosilicate bioactive glass 8 SrO.12CaO.8MgO.10B according to molar ratio2O3·4P2O5·58SiO2The raw materials of the components. Preparation thereofThe process is as follows: the temperature of the water bath kettle is firstly adjusted to 30 ℃. Cetyl ammonium bromide was dissolved in 100ml of deionized water, stirred for 10 minutes, added with ethyl orthosilicate, and stirred for 20 minutes. After adding 3ml of 25% aqueous ammonia, the mixture was stirred for 30 minutes. And adding tributyl borate and calcium nitrate tetrahydrate for reaction for 6 hours. The obtained product deionized water and absolute ethyl alcohol are alternately washed for 2 times. After drying, putting the powder into a muffle furnace, heating to 400 ℃ at the speed of 1 ℃ per minute, preserving heat for 6 hours, and then cooling along with the furnace. Thus obtaining the borosilicate bioactive glass with micro-nano level.
(2) Preparation of photosensitive silk fibroin solution:
dissolving photosensitive silk fibroin in water, and uniformly mixing to obtain a 30% photosensitive silk fibroin solution; 0.2% of a photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate was added.
(3) Preparing an ordered porous composite scaffold:
in order to make borosilicate bioactive glass uniformly disperse in photosensitive silk fibroin solution, firstly, dispersing borosilicate bioactive glass in 100 mu L of deionized water; and mixing the prepared borosilicate bioactive glass with the silk fibroin solution to prepare silk fibroin/bioglass slurry with the borosilicate bioactive glass content of 0.1%, and performing 3D printing to obtain the ordered porous composite scaffold.
The 3D printing comprises the following specific steps:
placing the slurry into a stereolithography Apparatus (SLA) printer, designing a 3D model through a computer, introducing into slicing software, processing, and storing as a stl (stereolithography) format file, wherein the thickness of the slice is set to be 0.05 mm; the exposure time was 15 s; the bottom exposure time was 60 s; the number of the bottom layers is 3; the lifting distance of the Z axis is 6 mm; the Z-axis lifting speed was 3 mm/s.
And printing the ordered porous composite material according to a pre-designed model and corresponding parameters.
(4) And placing the prepared ordered porous composite material in a photocuring instrument for photocuring and crosslinking for 1min to obtain an ordered porous composite scaffold product.
The prepared ordered porous composite material can be applied to bone tissue engineering and dental tissue engineering.
Specifically, the ordered porous composite material can be used for preparing a bracket and a composite artificial bone.
Example 9
(1) Preparation of borosilicate bioactive glass:
weighing borosilicate bioactive glass 8 SrO.12CaO.8MgO.10B according to molar ratio2O3·4P2O5·58SiO2The raw materials of the components. The preparation process comprises the following steps: the temperature of the water bath kettle is firstly adjusted to 50 ℃. Cetyl ammonium bromide was dissolved in 100ml of deionized water, stirred for 10 minutes, added with ethyl orthosilicate, and stirred for 20 minutes. After adding 2ml of 25% aqueous ammonia, the mixture was stirred for 30 minutes. And adding tributyl borate and calcium nitrate tetrahydrate for reaction for 6 hours. The obtained product deionized water and absolute ethyl alcohol are alternately washed for 4 times. After drying, putting the powder into a muffle furnace, heating to 450 ℃ at the speed of 1 ℃ per minute, and cooling along with the furnace after keeping the temperature for 8 hours. Thus obtaining the borosilicate bioactive glass.
(2) Preparation of photosensitive silk fibroin solution:
dissolving photosensitive silk fibroin in water, and uniformly mixing to obtain a 30% photosensitive silk fibroin solution; 0.3% of a photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate was added.
(3) Preparing an ordered porous composite scaffold:
in order to uniformly disperse borosilicate bioactive glass in a photosensitive silk fibroin solution, micro-nano borosilicate bioglass can be firstly dispersed in 300 mu L deionized water; mixing the prepared borosilicate bioactive glass with the silk fibroin solution to prepare silk fibroin/bioglass slurry with the content of the micro-nano borosilicate bioactive glass being 10%, and performing 3D printing to obtain the ordered porous composite scaffold.
The 3D printing comprises the following specific steps:
placing the slurry into a stereolithography Apparatus (SLA) printer, designing a 3D model through a computer, introducing into slicing software, processing, and storing as a stl (stereolithography) format file, wherein the thickness of the slice is set to be 0.2 mm; the exposure time was 20 s; the bottom exposure time was 80 s; the number of the bottom layers is 6; the lifting distance of the Z axis is 8 mm; the Z-axis lifting speed was 7 mm/s.
And printing the ordered porous composite material according to a pre-designed model and corresponding parameters.
(4) And placing the prepared ordered porous composite material in a photocuring instrument for photocuring and crosslinking for 1min to obtain an ordered porous composite scaffold product.
The prepared ordered porous composite material can be applied to bone tissue engineering and dental tissue engineering.
Specifically, the ordered porous composite material can be used for preparing a bracket and a composite artificial bone.
Example 10
(1) Preparation of borosilicate bioactive glass:
weighing borosilicate bioactive glass 8 SrO.12CaO.8MgO.10B according to molar ratio2O3·4P2O5·58SiO2The raw materials of the components. The preparation process comprises the following steps: the temperature of the water bath kettle is firstly adjusted to 40 ℃. Cetyl ammonium bromide was dissolved in 100ml of deionized water, stirred for 5 minutes, added with ethyl orthosilicate, and stirred for 10 minutes. After adding 5ml of 25% aqueous ammonia, the mixture was stirred for 30 minutes. And adding tributyl borate and calcium nitrate tetrahydrate for reaction for 6 hours. The obtained product deionized water and absolute ethyl alcohol are alternately washed for 6 times. After drying, putting the powder into a muffle furnace, heating to 500 ℃ at the speed of 1 ℃ per minute, preserving the temperature for 10 hours, and then cooling along with the furnace. Thus obtaining the borosilicate bioactive glass.
(2) Preparation of photosensitive silk fibroin solution:
dissolving photosensitive silk fibroin in water, and uniformly mixing to obtain a 30% photosensitive silk fibroin solution; then 1% of photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphonate was added.
(3) Preparing an ordered porous composite scaffold:
dispersing borate silicate bioglass in 800 mu L of deionized water; and mixing the prepared borosilicate bioactive glass with the silk fibroin solution to prepare silk fibroin/bioglass slurry with the borosilicate bioactive glass content of 8%, and performing 3D printing to obtain the ordered porous composite scaffold.
The 3D printing comprises the following specific steps:
placing the slurry into a stereolithography Apparatus (SLA) printer, designing a 3D model through a computer, introducing into slicing software, processing, and storing as a stl (stereolithography) format file, wherein the thickness of the slice is set to be 0.1 mm; the exposure time was 15 s; the bottom exposure time was 50 s; the number of the bottom layers is 4; the lifting distance of the Z axis is 6 mm; the Z-axis lifting speed was 4 mm/s.
And printing the ordered porous composite material according to a pre-designed model and corresponding parameters.
(4) And placing the prepared ordered porous composite material in a photocuring instrument for photocuring and crosslinking for 1min to obtain an ordered porous composite scaffold product.
The prepared ordered porous composite material can be applied to bone tissue engineering and dental tissue engineering.
Specifically, the ordered porous composite material can be used for preparing a bracket and a composite artificial bone.
Example 11
This embodiment is different from embodiment 7 in that: in this example, the slurry was placed in a mold and photo-crosslinked for curing, without using 3D printing technology.
Experimental analysis for degradation of two-ordered porous composite scaffold
And taking the ordered composite porous scaffold prepared in the example 7, namely 1BG/Sli-MA, 2BG/Sli-MA, 3BG/Sli-MA, 4BG/Sli-MA, 5BG/Sli-MA and 6 BG/Sli-MA. Weighing the ordered composite porous scaffold (initial mass w1), soaking in SBF for 14d, taking out the scaffold every 3 days, freezing in a refrigerator at-80 ℃ until the scaffold is uniformly freeze-dried for 3d after 14 days, and measuring the weight of the scaffold (w 2). The weight loss of the ordered composite porous scaffold is equal to the difference between the weights w2 and w1 after lyophilization. As can be seen from fig. 4, the addition of borosilicate bioactive glass affects the weight loss of the scaffold and increases with increasing borosilicate bioactive glass content, probably because it affects the degree of cross-linking of the original scaffold. Therefore, there is a more practical need to control the degradation rate of the scaffold by different borosilicate bioactive glass contents.
Experimental analysis of cytotoxicity of three-ordered porous composite scaffold
The ordered porous composite scaffold prepared in example 7, 1BG/Sli-MA, 2BG/Sli-MA, 3BG/Sli-MA, 4BG/Sli-MA, 5BG/Sli-MA and 6BG/Sli-MA, was sampled at a size of 2cm × 2cm square, and a Blank culture solution (Blank control) and silk fibroin (Sf) without borosilicate bioactive glass were used as a control, and the sample size of the silk fibroin control was also a sample at a size of 2cm × 2cm square. All cytotoxicity tests were performed according to GB/T16886.5-2003 selection leach liquor test method, according to cck8 instructions. The specific process is as follows:
(1) first, cell culture is performed: taking periodontal ligament stem cells (PDLSCs) and periodontal ligament stem cells to perform resuscitation-culture-passage-culture process, and digesting the cells for standby when the cells are transmitted to the third generation (5-6 days are needed under normal state).
(2) Secondly, preparing a leaching solution: calculating the conversion relationship between the weight and surface area of the bone cement samples of the experimental group and the control group by adopting a leaching liquor test method, and leaching in a leaching proportion of 6cm by adopting a-MEM cell culture solution (containing 15% fetal calf serum)2The leaching liquor of the experimental group and the control group is prepared at 37 ℃ for 24 h.
The prepared density is 1 multiplied by 103Inoculating 100ml of cell suspension into a 96-well plate, setting a blank group (only cell culture solution without leaching liquor), an experimental group and a control group, inoculating at least 3 holes into each group, culturing at 37 ℃ for 24h under the condition of containing 5% of carbon dioxide, discarding the culture solution, exchanging the blank group with the cell culture solution, and exchanging the experimental group and the control group with respective leaching liquor respectively. Culturing at 37 deg.C for 72h in 5% carbon dioxide incubator, adding cck-8, culturing for 1h, and measuring absorbance at 450nm with microplate reader. Relative proliferation rate (RGR) was calculated using the absorbance of the blank group as a standard, and the level of cytotoxic reaction of the samples of the experimental group and the control group was judged based on the RGR. The results of the cytotoxicity tests at 1, 3, 5 and 7 days are shown in FIG. 5,compared with the control group, the experimental group has more than 75% except 5%, 6% and 7 days which are less than 75%, which shows that the ordered porous composite scaffold of all the components has good biocompatibility.
Experimental analysis of the adhesion of the four periodontal ligament stem cells on the surface of the ordered porous composite scaffold
4 of the ordered porous composite scaffolds 3BG/Sli-MA prepared in example 7 (size 1 cm. times.1 cm) were taken (parallel experiments) and sterilized for use. Selecting 3 generation cells of periodontal ligament stem cells (PDLSCs), inoculating 100ul of cell suspension onto the surface of the stent, pre-adhering for 2 hours, pouring into a culture medium, changing the culture medium every 3 days, and culturing for 3 days and 7 days respectively. And taking out the 3d group, performing gradient dehydration, freeze drying for 1d, then shooting SEM, and observing the 7d group under a fluorescence microscope after dyeing according to the live-dead dyeing instruction. As can be seen from fig. 6, the 3d group of electron microscopy (see fig. 6(a), (b)) showed that the periodontal ligament stem cells (PDLSCs) cells spread out, stretching out the pseudopodia. In the 7d group, more viable cells were observed under a fluorescence microscope, and there were almost no red dead cells (see FIGS. 6(c), (d)).
Experimental analysis of osteogenic differentiation performance of PDLSCs promoted by five-ordered porous composite scaffold
The ordered porous composite scaffolds prepared in example 7, 1BG/Sli-MA, 3BG/Sli-MA and 5BG/Sli-MA, were used as controls with a Blank culture solution (Blank control) and silk fibroin (Sf) without borosilicate bioactive glass. Periodontal ligament stem cells (PDLSCs) were plated at 1 × 10 per well5Inoculating the culture medium to a 24-well plate, replacing the plate with control after 24h of adherence, and continuously culturing the culture medium in the bracket extract. The medium was changed every 2-3 days. ALP staining was used to quantitatively determine the effect of scaffolds on osteogenic differentiation of periodontal ligament stem cells (PDLSCs). ALP staining: after 7d and 14d of culture, the culture is stopped, PBS is washed for 3 times, 4% PFA is fixed for 30 minutes, PBS is rinsed for 3 times, and the world is stained by using Biyuntian ALP and the ALP secretion of periodontal ligament stem cells (PDLSCs) is detected. The specific steps refer to the ALP staining kit specification, the color development liquid is added once to incubate for 15 minutes in a dark place, and then the observation is carried out under an inverted microscope to take a picture. From fig. 7 it can be seen that silk fibroin and blank composition have comparable osteogenic activity. After the borosilicate bioactive glass is introduced, the content of the borosilicate bioactive glass can be obviously increasedAnd (3) osteogenic activity. However, the micro-nano borosilicate bioactive glass has certain inhibition on osteogenesis after being introduced too much.
In conclusion, the invention provides an ordered porous composite scaffold, and a preparation method and application thereof. According to the ordered porous composite scaffold provided by the invention, the borosilicate bioactive glass for inducing bone regeneration is added on the basis of the photosensitive silk fibroin, the performances of the borosilicate bioactive glass and the photosensitive silk fibroin are integrated, and the prepared ordered porous composite scaffold has good bioactivity, biodegradability and biocompatibility and can be used as a composite artificial bone integrating bone and alveolar bone repair and treatment. Meanwhile, after the porous borosilicate composite scaffold is used for alveolar bone repair, borosilicate bioactive glass can be gradually degraded to form a calcium-phosphorus compound in situ, so that the ordered porous composite scaffold provided by the invention has bioactivity and degradability. In addition, the borosilicate bioactive glass can release elements such as calcium (Ca), magnesium (Mg), silicon (Si) and the like which are beneficial to bone formation and blood vessel formation in the degradation process, so that the ordered porous composite scaffold provided by the invention can effectively induce defective vascularization and new bone regeneration, and rapidly repair bone loss injury caused by periodontitis in situ, thereby achieving a better effect on promoting bone tissue regeneration and repair. In addition, due to the introduction of borosilicate bioactive glass, compared with the traditional silk fibroin sponge dressing, the boron (B) precipitated from the ordered porous composite scaffold provided by the invention can regulate and control the bone loss caused by immunosuppression periodontitis, and a local slightly-alkaline environment is formed at the same time, so that the scaffold can effectively resist the invasion of acidophilic bacteria and has certain antibacterial performance.
The preparation method of the ordered porous composite scaffold provided by the invention adopts a silk fibroin in-situ polymerization method to uniformly disperse borosilicate bioactive glass in silk fibroin to obtain the ordered porous composite scaffold. The method is simple and easy to operate, can be formed at room temperature and is convenient to apply, and the prepared ordered porous composite scaffold has good biodegradability, biocompatibility and osteoinductive activity.
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 (23)
1. An ordered porous composite comprising photosensitive silk fibroin and bioactive glass dispersed in the photosensitive silk fibroin.
2. The ordered porous composite material of claim 1, wherein the photosensitive silk fibroin is modified by photopolymerization chemical crosslinking reaction of silk fibroin and glycidyl methacrylate.
3. The ordered porous composite of claim 1, wherein the bioactive glass comprises 0.1-10% by weight of the ordered porous composite.
4. The ordered porous composite of claim 2, wherein the bioactive glass comprises 1-8% by weight of the ordered porous composite, preferably 3-6%.
5. The ordered porous composite of claim 1, wherein the bioactive glass is a borosilicate bioactive glass.
6. The ordered porous composite of claim 5, wherein the borosilicate bioactive glass is selected from one or more of the following compositions: aXO bB2O3·cP2O5·dSiO2；
Wherein, a, b, c and d are mole fractions, a is 20-66, b is 2-48, c is 2-10 and d is 30-76; x is Ca and/or Mg and/or Sr.
7. The ordered porous composite of claim 6, wherein the borosilicate bioactive glass comprises one or at least two of the following compositions:
10SrO·10CaO·10MgO·40B2O3·2P2O5·28SiO2、
20CaO·8MgO·27B2O3·4P2O5·41SiO2、
14SrO·20CaO·36B2O3·2P2O5·28SiO2、
14SrO·8CaO·8MgO·40B2O3·2P2O5·28SiO2、
8SrO·12CaO·8MgO·10B2O3·4P2O5·58SiO2、
14SrO·8CaO·8MgO·38B2O3·2P2O5·30SiO2、
10SrO·10CaO·10MgO·38B2O3·2P2O5·30SiO2、
20CaO·8MgO·27B2O3·4P2O5·41SiO2、
14SrO·18CaO·36B2O3·2P2O5·30SiO2、
20CaO·8MgO·30B2O3·4P2O5·38SiO2、
20CaO·20B2O3·2P2O5·58SiO2。
8. the ordered porous composite of claim 1 or 5, wherein the bioactive glass has a particle size in the range of 30-300 nm.
9. The ordered porous composite of claim 5, wherein the borosilicate bioactive glass further comprises ZnO, Ag2O, CuO and CeO2One or at least two of them.
10. The ordered porous composite of claim 9, wherein the ZnO is present in an amount of 0-2% by mole and Ag is present in an amount of20 to 0.5 percent of O, 0 to 0.5 percent of CuO and CeO2The molar content of (A) is 0-1%.
11. The ordered porous composite of claim 1, wherein the ordered porous composite has a pore size of 50-300 μ ι η, preferably 70-150 μ ι η; the ordered porous composite has a pore density of 50 to 300ppi, preferably 80 to 280ppi, more preferably 100-260 ppi.
12. A method for preparing an ordered porous composite according to any of claims 1 to 4, comprising the steps of: mixing the photosensitive silk fibroin solution with bioactive glass to obtain slurry; and carrying out photocuring molding on the obtained slurry to obtain the ordered porous composite material.
13. The method of claim 12, wherein the bioactive glass comprises 0.1-10% by weight of the ordered porous composite.
14. The method of preparing an ordered porous composite material according to claim 12, wherein said bioactive glass is a borosilicate bioactive glass.
15. The method for preparing the ordered porous composite material according to claim 12, wherein the specific method for photocuring molding is as follows: and (3) placing the slurry into a photocuring three-dimensional forming printer, printing according to a preset model and parameters, and simultaneously carrying out photocuring curing.
16. The method for preparing the ordered porous composite material according to claim 12, wherein the specific method for photocuring molding is as follows: and (3) placing the slurry in a mold, and carrying out photo-crosslinking curing.
17. The method of claim 12, wherein the photosensitive silk fibroin solution is an aqueous solution of photosensitive silk fibroin.
18. The method of preparing an ordered porous composite according to claim 17, wherein the photosensitive fibroin solution contains a photoinitiator.
19. The method of preparing an ordered porous composite according to claim 18, wherein said photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate.
20. The method of making an ordered porous composite material according to claim 14, wherein said borosilicate bioactive glass is formed by at least the steps of:
s1: dissolving hexadecyl ammonium bromide in deionized water, adding tetraethoxysilane and ammonia water, and stirring to obtain a mixed solution;
s2, adding tributyl borate and calcium nitrate tetrahydrate into the mixed solution obtained in the step S1, and reacting to obtain coarse micro-nano borosilicate bioactive glass;
and S3, washing, drying, preserving heat and cooling the coarse micro-nano borosilicate bioactive glass obtained in the step S2 to obtain the nano borosilicate bioactive glass.
21. The method for preparing the ordered porous composite material of claim 14, wherein one or more of silver nitrate, copper sulfate and cerium nitrate is further added to the mixed solution before the reaction of step S2.
22. Use of an ordered porous composite according to any of claims 1 to 11 or prepared according to the method of any of claims 12 to 21 in bone tissue engineering and dental tissue engineering.
23. An artificial bone or scaffolds comprising an ordered porous composite material according to any one of claims 1-11 or prepared according to the method of any one of claims 12-21.
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