CN114272443B - Preparation method and application of zinc silicate nanoparticle composite fiber scaffold - Google Patents
Preparation method and application of zinc silicate nanoparticle composite fiber scaffold Download PDFInfo
- Publication number
- CN114272443B CN114272443B CN202111505370.7A CN202111505370A CN114272443B CN 114272443 B CN114272443 B CN 114272443B CN 202111505370 A CN202111505370 A CN 202111505370A CN 114272443 B CN114272443 B CN 114272443B
- Authority
- CN
- China
- Prior art keywords
- zinc silicate
- zinc
- scaffold
- nanoparticle composite
- fibers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention relates to a preparation method and application of a zinc silicate nanoparticle composite fiber scaffold. The zinc silicate nanoparticle composite fiber scaffold comprises: the biological polymer fiber and the zinc silicate nano-particles embedded in the biological polymer fiber; the zinc silicate nanoparticle composite fiber scaffold can promote the healing of skin wound and the reconstruction of skin neurovascular network at the same time.
Description
Technical Field
The invention relates to a preparation method of a zinc silicate nanoparticle composite fiber scaffold and application of the zinc silicate nanoparticle composite fiber scaffold in the regeneration of nervous skin, in particular to the application in the healing of skin wounds and the reconstruction of a neurovascular network thereof, belonging to the field of biological materials.
Background
Skin, which is the outermost organ tissue of the human body, is easily burned and scalded by flames, hot water, chemicals, or the like in daily life. Serious burns and scalds can not only damage the dermal matrix of the skin, but also damage the neurovascular network in the skin, so that the skin loses the ability of sensing the external environment, and the damaged neurovascular network is difficult to repair by self.
At present, the conventional wound dressing can only play a role in promoting wound healing, but does not have the capacity of promoting the reconstruction of a new skin neurovascular network, so that the effect of skin repair is limited. Therefore, the novel bioactive multifunctional wound dressing is developed, effectively promotes the reconstruction of a neurovascular network in the skin while promoting the healing of the wound, and has important significance for realizing functional skin tissue regeneration.
Prior art documents:
1.Lv F,Wang J,Xu P,et al.Aconducive bioceramic/polymer composite biomaterial for diabetic wound healing[J].Acta biomaterialia,2017:128-143.
2.Cheng L,Cai Z,Ye T,et al.Injectable Polypeptide-Protein Hydrogels for Promoting Infected Wound Healing[J].Advanced Functional Materials,2020,2001196.
3.Peng L,Xu X,Huang Y,et al.Self-Adaptive All-In-One Delivery Chip for Rapid Skin Nerves Regeneration by Endogenous Mesenchymal Stem Cells[J].Advanced Functional Materials,2020,30,2001751.
4.Yu Q,Han Y,Wang X,et al.Copper Silicate Hollow Microspheres-Incorporated Scaffolds for Chemo-Photothermal Therapy of Melanoma and Tissue Healing[J].Acs Nano,2018,12,2695-2707.
disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a multifunctional bioactive skin tissue engineering scaffold material which can effectively promote the healing of skin wound and promote the reconstruction of a neurovascular network of the skin.
In a first aspect, the present invention provides a zinc silicate nanoparticle composite fibrous scaffold comprising: the biological polymer fiber and the zinc silicate nano-particles embedded in the biological polymer fiber; the composite fiber scaffold can promote the healing of the skin wound and the reconstruction of the skin neurovascular network at the same time.
Preferably, the mass ratio of the zinc silicate nanoparticles to the biopolymer fibers is 2-40%.
Preferably, the biopolymer fibers are biocompatible polymer fibers, preferably polylactic acid, polycaprolactone or chitosan polymer fibers, and more preferably polycaprolactone polymer fibers; the diameter range of the biopolymer fibers is 0.4-2 μm.
Preferably, the zinc silicate nanoparticles have a spindle-shaped structure, a length range of 600-900 nm, a width range of 200-500 nm and a particle size distribution of 400-800 nm.
In a second aspect, the invention provides a preparation method of the zinc silicate nanoparticle composite fiber scaffold, which comprises the following steps: fully stirring and mixing a zinc salt solution, a silicon source solution and a sodium hydroxide solution to form a mixed solution, then carrying out hydrothermal reaction, collecting a precipitate and washing to obtain the zinc silicate nanoparticles; and uniformly mixing the zinc silicate nanoparticles and the biopolymer fiber material in a hexafluoroisopropanol solution, then filling the uniformly mixed solution into an injector for electrostatic spinning, and drying to obtain the zinc silicate nanoparticle composite fiber scaffold.
Preferably, the zinc salt is at least one of zinc nitrate, zinc nitrate hydrate, zinc sulfate hydrate or anhydrous zinc chloride, and the silicon source is sodium silicate hydrate or tetraethoxysilane TEOS; the mass concentration of the zinc salt in the mixed solution is 0.05-0.1 g/mL, the mass concentration of the sodium hydroxide is 0.2-0.4 g/mL, and the mass concentration of the silicon source is 0.01-0.1 g/mL.
Preferably, the temperature of the hydrothermal reaction is 160-240 ℃ and the time is 12-36 h.
Preferably, the mass ratio of the zinc silicate nanoparticles to the biopolymer fiber material in the hexafluoroisopropanol solution is (0.02-0.4): 1, preferably 0.1.
Preferably, the parameters of the electrostatic spinning are as follows: the spinning voltage is 8-12 kV, the rotation speed of the receiving roller is 1800-2500 r/min, the extrusion speed of the needle is 0.01-0.05 mL/min, and the distance between the receiving roller and the needle is 10-20 cm.
In a third aspect, the invention also provides application of the zinc silicate nanoparticle composite fiber scaffold in a bioactive skin tissue engineering scaffold material with dual functions of wound healing and nerve vascular network reconstruction.
Advantageous effects
The zinc silicate nanoparticle composite fiber scaffold provided by the invention has good biocompatibility and excellent in-vitro vascularization and nervogenesis activities, and provides a simple, convenient and effective scheme for realizing functional skin regeneration. Relevant animal experiments prove that the zinc silicate nanoparticle composite fiber scaffold can effectively promote the healing of the scald wound of the skin and accelerate the reconstruction of a neurovascular network in a damaged area.
Drawings
FIG. 1 is the silicic acid prepared in example 1Zinc nanoparticles (Zn) 2 SiO 4 ) A characterization map of the crystal phase, the particle size and the microstructure of (a);
FIG. 2 is a micro-topography characterization of the composite nanofiber scaffold (PCL +5 ZS) prepared in example 1;
FIG. 3 is a representation of the microstructure and elemental distribution of fibrous scaffolds prepared in examples 1-3 and comparative example 1;
FIG. 4 is a graph of fiber diameter distribution of the fiber scaffolds prepared in examples 1-3 and comparative example 1;
FIG. 5 is a graph showing the in vitro proliferation and migration of endothelial cells of the fibrous scaffolds prepared in examples 1-3 and comparative example 1;
FIG. 6 is a graphical representation of the in vitro pro-angiogenesis of fibrous scaffolds prepared in examples 1-3 and comparative example 1;
FIG. 7 is a characterization of the in vitro proneurization of fibrous scaffolds prepared in examples 1-3 and comparative example 1;
FIG. 8 is a schematic diagram showing the experimental effect of the treatment of skin scald in vivo in a white mouse;
fig. 9 is a representation of neurovascular reconstruction of new skin.
Detailed Description
The present invention is further illustrated by the following examples and examples, with the understanding that the following examples are intended to be illustrative of the invention only and are not intended to be limiting.
The invention provides a zinc silicate nanoparticle composite fiber scaffold, which comprises: the biological polymer fiber and the zinc silicate nano-particles embedded in the biological polymer fiber. The nanoparticles can be embedded in the biopolymer fibers in a manner of being completely or partially embedded in the fibers, and/or partially exposed outside the fibers, and the like.
The mass ratio of the zinc silicate nano-particles to the biopolymer fibers is 2-40%. An excessively low content of zinc silicate nanoparticles may result in poor bioactivity of the composite fiber scaffold, while an excessively high content of zinc silicate nanoparticles may be detrimental to the preparation of the composite fiber scaffold and may result in potential cytotoxicity. The scaffold can be used for healing skin wound and reconstructing a neurovascular network thereof.
The invention takes the zinc silicate nano-particles as the biological activator for the first time to promote the regeneration and reconstruction of the neurovascular network in the damaged skin. Bioactive zinc and silicon ions released by the zinc silicate nano particles are utilized to stimulate nerves and blood vessels, so that the reconstruction of a neurovascular network can be effectively promoted while the healing of a wound surface is promoted. Because the nanoparticles cannot cover the wound surface and are not suitable for directly using the nanoparticles in skin repair, the zinc silicate nanoparticles are embedded into the biopolymer fibers, so that the fibrous scaffold has the capabilities of promoting wound surface healing and reconstructing a neurovascular network.
The angiogenesis activity of the zinc silicate nanoparticles is proved by evaluating the effects of endothelial cell proliferation, migration, in vitro angiogenesis-related gene expression and in vivo angiogenesis; the nerve regeneration capacity of the zinc silicate nanoparticles is proved by evaluating the proliferation of Schwann cells, the expression of genes related to neurotrophic factors in vitro and the effect of nerve regeneration in vivo. The zinc silicate nanoparticle composite fiber scaffold can effectively promote wound healing and neurovascular network reconstruction, and provides a simple and effective scheme for realizing functionalized skin regeneration.
The biopolymer fibers are fibers made of a biopolymer, and the fiber diameter is in the range of 0.4 to 2 μm. The biopolymer can be a polymer material with good biocompatibility, such as polylactic acid, polycaprolactone, chitosan, and the like, and in some preferred embodiments, the biopolymer is polycaprolactone. The fiber scaffold is a three-dimensional network structure consisting of directionally arranged nano fibers, and the fiber diameter of the fiber scaffold is not influenced by the addition of the zinc silicate nano particles.
In some embodiments, the zinc silicate nanoparticles are a spindle-shaped structure formed by self-assembly stacking of a plurality of zinc silicate nano short rods, the length of the zinc silicate nanoparticles ranges from 600 nm to 900nm, the width of the zinc silicate nanoparticles ranges from 200 nm to 500nm, and the particle size distribution of the zinc silicate nanoparticles ranges from 400nm to 800nm. The spindle-shaped zinc silicate nanoparticles have excellent angiogenesis activity and neurogenesis activity, can promote healing of a scalded skin wound surface, and can reconstruct a neurovascular network in damaged skin tissues.
The zinc silicate nanoparticle composite fiber scaffold can be prepared by embedding the zinc silicate nanoparticles onto the biopolymer fibers by utilizing an electrostatic spinning technology. Specifically, the fiber scaffold is obtained by uniformly mixing zinc silicate nanoparticles with a biopolymer solution and then performing electrostatic spinning. Simple process, batch production and stable material performance. The specific preparation method mainly comprises the following two steps.
Step (1): and (3) preparing zinc silicate nanoparticles. Sequentially adding zinc salt (zinc nitrate and/or Zn (NO) hydrate thereof) 3 ) 2 ·6H 2 ZnSO, zinc sulfate and/or hydrate thereof 4 ·7H 2 O, anhydrous zinc chloride ZnCl 2 Etc.) solution and sodium hydroxide solution are added to a silicon source (which may be sodium silicate hydrate Na) 2 SiO 3 ·9H 2 O, tetraethyl orthosilicate TEOS, etc.) solution, and then carrying out hydrothermal reaction after fully stirring and mixing. Wherein, the mass concentration of the zinc salt in the mixed solution is controlled to be 0.05-0.1 g/mL, the mass concentration of the sodium hydroxide is controlled to be 0.2-0.4 g/mL, the mass concentration of the silicon source is controlled to be 0.01-0.1 g/mL, the temperature of the hydrothermal reaction can be 160-240 ℃, and the time of the hydrothermal reaction can be 12-36 h. And collecting white precipitate after the reaction is finished, and washing to obtain the zinc silicate nano particles.
Step (2): preparing the zinc silicate nanoparticle composite fiber scaffold. Adding the zinc silicate nanoparticles prepared in the step (1) into a hexafluoroisopropanol solution, and performing ultrasonic dispersion for 30min to obtain the hexafluoroisopropanol solution containing the zinc silicate nanoparticles (the concentration of the zinc silicate nanoparticles is 0.2-4 wt.%). And then adding biopolymer fibers such as polycaprolactone materials, controlling the concentration of the biopolymer fibers to be 5-20 wt.%, preferably 10wt.%, and controlling the mass ratio of the zinc silicate nanoparticles to the biopolymer fibers to be (0.02-0.4): 1, preferably 0.1. After the biopolymer fiber material was fully dissolved, the mixed solution was put into a 5-20 mL syringe for electrospinning. The type of the electrostatic spinning needle is 16-26G, the spinning voltage is 8-12 kV, the rotating speed of the receiving roller is 1800-2500 r/min, the extruding speed of the needle is 0.01-0.05 mL/min, and the distance between the receiving roller and the needle is 10-20 cm. And after the electrostatic spinning is finished, drying the obtained product for 12 to 48 hours at room temperature in a fume hood to obtain the zinc silicate nanoparticle composite fiber scaffold.
In an optional embodiment, the zinc silicate nanoparticles prepared by a hydrothermal method can also be added into a hexafluoroisopropanol solution containing a biopolymer fiber material such as polycaprolactone and the like for electrostatic spinning, so as to obtain the zinc silicate nanoparticle composite fiber scaffold.
The present invention will be described in detail by way of examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art may be made in light of the above teachings. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.
Example 1
Step (1): and (3) preparing zinc silicate nanoparticles. 1.19g of Zn (NO) 3 ) 2 ·6H 2 O and 0.57g of Na 2 SiO 3 ·9H 2 And dissolving O in 5mL of deionized water to form a zinc nitrate solution and a sodium silicate solution respectively, dropwise adding the zinc nitrate solution into the sodium silicate solution and fully stirring for 5min, and then dropwise adding 6mL of sodium hydroxide solution (1 mol) into the mixed solution and fully stirring for half an hour. And then, putting the solution into a 25mL hydrothermal kettle, reacting at 220 ℃ for 24 hours, collecting white precipitate, and washing with deionized water and absolute ethyl alcohol to obtain the zinc silicate nanoparticles.
Step (2): preparing the zinc silicate nanoparticle composite fiber scaffold. 0.05g of the zinc silicate nanoparticles prepared in step (1) was weighed and dispersed in 10mL of hexafluoroisopropanol solution, and then 1g of polycaprolactone material (PCL, molecular weight 80000, available from Sigma-Aldrich chemical reagent Co.) was added, and after being sufficiently dissolved by magnetic stirring, it was put into a 10mL syringe, and the syringe was put on the pusher of an electrospinning machine to conduct electrospinning. The type of the needle of the electrostatic spinning injector is 20G, the spinning voltage is 10kV, the rotating speed of a receiving roller is 2200r/min, the extruding speed of the needle is 0.02mL/min, and the distance between the receiving roller and the needle is 15cm. And after the electrostatic spinning is finished, drying the composite nanofiber scaffold in a fume hood at room temperature for 24 hours to obtain the composite nanofiber scaffold (PCL +5 ZS).
FIG. 1 shows zinc silicate nanoparticles (Zn) prepared in example 1 2 SiO 4 ) The crystal phase, the grain diameter and the microstructure of (2) are characterized. Wherein, the material comprises (a) a scanning electron microscope picture, (b) an XRD atlas, (c) a particle size distribution picture, (d) a transmission electron microscope picture, (e) a selected area electron diffraction picture and (f) a high-resolution transmission electron microscope picture. As can be seen from FIG. 1, the synthesized zinc silicate nanoparticles have a good morphology structure, a spindle-shaped morphology structure, a length of about 700nm, a width of about 400nm, and a particle size distribution range of 500-700 nm.
Fig. 2 is a micro-topography characterization of the composite nanofiber scaffold (PCL +5 ZS) prepared in example 1.
Example 2
Step (1): preparing zinc silicate nano particles. The same as in example 1.
Step (2): preparing the zinc silicate nano particle composite fiber scaffold. 0.1g of the zinc silicate nanoparticles prepared in step (1) was weighed and dispersed in 10mL of hexafluoroisopropanol solution, and then 1g of polycaprolactone material (PCL, molecular weight 80000, available from Sigma-Aldrich chemical Co.) was added, and after being dissolved sufficiently by magnetic stirring, it was loaded into a 10mL syringe, which was placed on the pusher of an electrospinning machine for electrospinning. The type of the needle of the electrostatic spinning injector is 20G, the spinning voltage is 10kV, the rotating speed of a receiving roller is 2200r/min, the extruding speed of the needle is 0.02mL/min, and the distance between the receiving roller and the needle is 15cm. And after the electrostatic spinning is finished, drying the composite nanofiber scaffold in a fume hood at room temperature for 24 hours to obtain the composite nanofiber scaffold (PCL +10 ZS).
Example 3
Step (1): preparing zinc silicate nano particles. The same as in example 1.
Step (2): preparing the zinc silicate nanoparticle composite fiber scaffold. 0.2g of the zinc silicate nanoparticles prepared in step (1) was weighed and dispersed in 10mL of hexafluoroisopropanol solution, and then 1g of polycaprolactone material (PCL, molecular weight 80000, available from Sigma-Aldrich chemical Co.) was added, and after being dissolved sufficiently by magnetic stirring, it was loaded into a 10mL syringe, which was placed on the pusher of an electrospinning machine for electrospinning. The type of the needle of the syringe for electrostatic spinning is 20G, the spinning use voltage is 10kV, the rotating speed of the receiving roller is 2200r/min, the extruding speed of the needle is 0.02mL/min, and the distance between the receiving roller and the needle is 15cm. And after the electrostatic spinning is finished, drying the obtained product in a fume hood at room temperature for 24 hours to obtain the composite nanofiber scaffold (PCL +20 ZS).
Comparative example 1
A pure polycaprolactone fiber scaffold was used as a control. 1g of polycaprolactone material (PCL, molecular weight 80000, available from Sigma-Aldrich chemical Co.) was dissolved in 10mL of hexafluoroisopropanol solution, and after being dissolved sufficiently by magnetic stirring, the solution was loaded into a 10mL syringe, and the syringe was placed on the pusher of an electrospinning machine to conduct electrospinning. The type of the needle of the electrostatic spinning injector is 20G, the spinning voltage is 10kV, the rotating speed of a receiving roller is 2200r/min, the extruding speed of the needle is 0.02mL/min, and the distance between the receiving roller and the needle is 15cm. And after the electrostatic spinning is finished, drying the obtained product in a fume hood at room temperature for 24 hours to obtain the pure nanofiber scaffold (PCL).
FIG. 3 is a representation of the microscopic morphology and elemental distribution of the fibrous scaffolds prepared in examples 1-3 and comparative example 1. Wherein (a) is the microscopic morphology of four groups of fiber scaffolds with different zinc silicate nanoparticle contents, which are sequentially PCL, PCL +5ZS, PCL +10ZS and PCL +20ZS, and (b-e) is the element distribution of Zn, si and O in the PCL +20ZS fiber scaffold. As can be seen from the figure, the composite fiber scaffold has a three-dimensional network formed by directionally arranged fibers, wherein zinc silicate particles are embedded or semi-coated on the fibers, and the zinc silicate particles are uniformly distributed on the fiber scaffold without agglomeration.
FIG. 4 is a graph showing fiber diameter distribution of the fiber scaffolds prepared in examples 1-3 and comparative example 1. It can be seen from the figure that the addition of zinc silicate nanoparticles does not affect the diameter distribution or mean diameter of the nanofibers.
CCK-8 (Cell counting Kit-8, dojindo, japan) was used to experimentally characterize the effect of the fibrous stents prepared in examples 1-3 and comparative example 1 on the endothelial Cell proliferation capacity in vitro. First, the fiber scaffolds were sterilized with 75% ethanol and washed with PBS solution, then placed in 48-well plates, endothelial cells were inoculated and cultured at a density of 500 cells/well, the solution was changed every other day, the culture medium containing 10% CCK-8 solution was added at preset 1, 3, and 5 days, and the cells were cultured in an incubator (37 ℃,5% CO) 2 ) And then measuring the absorbance of all samples at 450nm to evaluate the proliferation of the cells.
Transwell migration experiments were used to characterize the effect of the fibrous scaffolds prepared in examples 1-3 and comparative example 1 on endothelial cell migration ability in vitro. The sterilized fibrous scaffold was placed in a 24-well plate, a Transwell chamber with a pore diameter of 8 μm was placed in the plate, endothelial cells were seeded at a density of 5000 cells/well in the upper chamber of the Transwell, cells were replaced with serum-free medium for 12 hours after cell adhesion, then cells were fixed with 4% paraformaldehyde and non-migrated cells in the upper chamber were erased with a cotton swab, and cells migrated to the lower surface of the chamber were stained with 0.1% of crytal violet and photographed under a microscope and statistically analyzed.
FIG. 5 is a characterization chart of in vitro endothelial cell proliferation and migration promotion of fibrous scaffolds prepared in examples 1-3 and comparative example 1. Wherein, (a) is the proliferation result of endothelial cells cultured for 1, 3, 5 days, (b) is the migration number statistics of endothelial cells, and (c) is the optical photograph of endothelial cell migration. The CCK-8 analysis result proves that the added zinc silicate nano-particles have no cytotoxicity to endothelial cells, and the composite fiber scaffold has excellent biocompatibility. Subsequently, from the results of Transwell cell migration experiments, it can be found that the fibrous scaffold containing a certain amount of zinc silicate nanoparticles has a significant promotion effect on the migration of endothelial cells, and further proves the excellent biological activity of the fibrous scaffold.
The influence of the fiber scaffold prepared in examples 1-3 and comparative example 1 on endothelial cell differentiation in vitro was characterized by using RT-qPCR (Real-time quantitative polymerase chain reaction) experiments, placing the sterilized fiber scaffold in a 12-well plate, performing inoculation culture on endothelial cells at a density of 50000 cells/well, changing the solution every other day, extracting total RNA of each group of cells by using Trizol reagent after 5 days of culture, performing reverse transcription on the RNA into cDNA by using a ReverTra Ace-alpha kit, and exploring the gene expression by using SYBR Green fluorescent Real-time quantitative PCR.
FIG. 6 is a graphical representation of the in vitro pro-angiogenesis of fibrous scaffolds prepared in examples 1-3 and comparative example 1. Wherein (a-d) is the results of the in vitro angiogenesis promoting related genes of the fiber scaffolds prepared in examples 1-3 and comparative example 1, and are the gene expressions of VEGF, eNOS, KDR, VE-cad in endothelial cells, respectively, and it can be seen that the zinc silicate nanoparticle composite fiber scaffold can significantly up-regulate the expressions of the angiogenesis related genes of HUVECs, VEGF, eNOS, KDR, VE-cad, indicating that zinc and silicon ions released by the zinc silicate composite fiber scaffold have certain in vitro angiogenesis promoting capability.
The influence of the fiber scaffolds prepared in example 1 and comparative example 1 on the expression of endothelial cell vascularization related proteins in vitro was characterized by using immunofluorescence protein staining experiments, placing the sterilized fiber scaffolds in 12-well plates, performing inoculation culture on endothelial cells for 5 days according to the density of 50000 cells/well, and changing the solution every other day. Firstly, fixing cells by using 4% paraformaldehyde, then carrying out treatments such as sealing, primary antibody and secondary antibody incubation, cytoskeleton and cell nucleus staining and the like on the cells, and finally photographing by using a confocal microscope and carrying out statistical analysis.
Fig. 6 shows the results of in vitro endothelial cell CD31 protein expression promotion of the fibrous scaffold prepared in example 1 and comparative example 1 in (e) and (f) in fig. 6, wherein (e) is the statistical result of the mean fluorescence intensity of the endothelial cell CD31 protein staining and (f) is a photograph of the endothelial cell CD31 protein staining. It can be seen that PCL +5ZS showed significantly high expression of CD31 protein compared to the PCL control group, indicating its excellent angiogenic ability.
CCK-8 (Cell counting Kit-8, dojindo, japan) was used to experimentally characterize the effect of the fiber scaffold prepared in examples 1-3 and comparative example 1 on the proliferation capacity of Schwann cells in vitro. The fiber scaffolds were first sterilized with 75% alcohol and washed with PBS solution, placed in 48-well plates, and the resulting plates were washedInoculating and culturing Schwann cells at a density of 500 cells/well, changing the medium every other day, adding culture medium containing 10% CCK-8 solution at predetermined 1, 3, and 5 days, and adding CO in the culture box (37 deg.C, 5% 2 ) And then measuring the absorbance of all samples at 450nm to evaluate the proliferation of the cells.
RT-qPCR (Real-time quantitative polymerase chain reaction) experiments were used to characterize the effect of the fiber scaffolds prepared in examples 1-3 and comparative example 1 on Schwann cell differentiation in vitro. Placing the sterilized fiber scaffold in a 12-hole plate, inoculating and culturing Schwann cells according to the density of 50000 cells/hole, changing liquid every other day, extracting total RNA of each group of cells by using a Trizol method after culturing for 5 days, carrying out reverse transcription on the RNA into cDNA by using a ReverTra Ace-alpha kit, and exploring the gene expression condition by using a SYBR Green fluorescent real-time quantitative PCR method.
FIG. 7 is a graphical representation of the in vitro proneurization of the fibrous scaffolds prepared in examples 1-3 and comparative example 1. Wherein (a-e) is a characteristic diagram of the fiber scaffold prepared in examples 1-3 and comparative example 1 for promoting the proliferation and differentiation of Schwann cells in vitro, (a) is the proliferation result of Schwann cells cultured for 1, 3 and 5 days, and (b-e) is the gene expression of NCAM, BDNF, PMP22 and NGF in the Schwann cells respectively, and the CCK-8 analysis result proves that the added zinc silicate nanoparticles have no obvious cytotoxicity on SCs, which indicates that the nanofiber scaffold has excellent biocompatibility. In addition, the zinc silicate nanoparticle composite fiber scaffold can remarkably up-regulate the expression of SCs nerve regeneration related genes NCAM, BDNF, PMP22 and NGF, and the zinc and silicon ions released by the zinc silicate composite fiber scaffold have certain in-vitro nerve regeneration promoting capacity.
The effect of the fiber scaffolds prepared in example 1 and comparative example 1 on the expression of schwann cell specific protein in vitro was characterized by using immunofluorescence protein staining experiments, placing the sterilized fiber scaffolds in 12-well plates, inoculating and culturing the schwann cells at a density of 50000 cells/well for 5 days, and changing the solution every other day. Firstly, fixing cells by using 4% paraformaldehyde, then sealing the cells, incubating the cells with a primary antibody and a secondary antibody, and after staining cytoskeleton and cell nucleus, photographing by using a confocal microscope and carrying out statistical analysis.
In fig. 7, (f) and (g) are the results of the in vitro snowwann cell S100 protein expression promotion of the fiber scaffold prepared in example 1 and comparative example 1, wherein (f) is the statistical result of the mean fluorescence intensity of the snowwann cell S100 protein staining, and (g) is the photograph of the S100 protein staining of the snowwann cells. It can be seen that PCL +5ZS showed significantly high expression of S100 protein compared to PCL control, indicating its excellent neurogenesis activity.
The fibrous scaffolds prepared in example 1 and comparative example 1 were used to characterize the effects of promoting the healing of skin wounds and the reconstruction of a neurovascular network using a mouse skin scald experiment. The method comprises the steps of firstly carrying out intraperitoneal injection anesthesia by using sodium pentobarbital, then removing back hairs by using a shaver, disinfecting back skin by using 75% alcohol, pressing an iron rod with the diameter of 10mm at 100 ℃ on the back of a white mouse for 5 seconds to create a scald wound surface with the diameter of 10mm, then attaching a fibrous support to the wound surface and fixing by using 3M glue, wherein only one layer of 3M glue is fixed when the Blank group is not treated. The fibrous scaffolds were replaced every two days and photographed records were taken at 0, 2, 5, 8, 11 and 14 days after surgery.
At day 8 and 14, wound tissue was taken for subsequent histological analysis. Skin tissues were fixed with 4% paraformaldehyde, then embedded in dehydrated paraffin, and then cut into 7 μm pieces. The wound healing effects such as re-epithelialization and the like of the slices were analyzed by staining the slices with hematoxylin-eosin reagent (H & E, beyotime, china), and recorded by photographing under a microscope.
Fig. 8 is a schematic diagram of the experimental effect of the treatment of skin scald of mice in vivo. The method comprises the following steps of (a) photographing wound surfaces at different time points, (b) counting wound closure rate at different time points, (c) drawing the sizes of the wound surfaces at different time points, and (d) obtaining HE staining results of the wound surfaces on 8 th and 14 th days. The statistical result of the wound area size in the treatment process shows that the PCL +5ZS nanofiber group can effectively promote the scald wound healing compared with a PCL pure nanofiber group and a blank group. In addition, by performing histological staining analysis on the skin samples on day 8 and day 14, it can be seen from hematoxylin-eosin (H & E) staining results that the skin wound of the PCL +5ZS nanofiber group had completely healed on day 14, more dermal matrix deposition occurred, and good re-epithelization was shown, while the blank group and the PCL control group still had some scab residues and the wound healing was not complete, indicating that the zinc silicate nanoparticle composite fiber scaffold effectively promoted the healing of the skin scald wound of the mice by accelerating the dermal matrix deposition and re-epithelization.
The effect of the fibrous scaffolds prepared in example 1 and comparative example 1 on the promotion of the skin wound neurovascular network reconstruction was characterized using an immunofluorescent protein staining experiment. On day 8 and 14, wound tissue was taken, and skin tissue was fixed with 4% paraformaldehyde, then embedded in dehydrated paraffin, and then cut into 7 μm pieces. Blood vessels and nerve fibers of the newborn skin were stained with a CD31 (Abcam, UK) dye and a PGP9.5 (Servicebio, china) dye, followed by photographic recording using a confocal microscope.
Fig. 9 is a neurovascular reconstruction characterization of neonatal skin. Wherein, (a) is the result of blood vessel CD31 staining on the wound surface at the 8 th day and the 14 th day, (b) is the statistical result of new blood vessel CD31 staining on the 8 th day and the 14 th day, (c) is the result of nerve fiber PGP9.5 staining on the wound surface at the 8 th day and the 14 th day, and (d) is the statistical result of nerve fiber PGP9.5 staining on the wound surface at the 8 th day and the 14 th day. As can be seen from the CD31 immunofluorescent staining results, the number of CD31 positive expressions of the PCL +5ZS nanofiber group was significantly higher on day 8 and 14 than the other two groups. In addition, the statistical result of the new blood vessels also shows that the addition of the zinc silicate nano-particles has obvious promotion effect on the blood vessel regeneration. From the immunofluorescence staining result of PGP9.5 nerve fibers, the positive expression area of PGP9.5 of the PCL +5ZS nanofiber group is obviously higher than that of the other two groups on the 8 th day and the 14 th day, the statistical result of the area of the nerve fibers on the wound surface also shows that the addition of the zinc silicate nanoparticles has an obvious promotion effect on nerve regeneration, and the zinc silicate nanoparticle composite fiber scaffold can effectively promote the regeneration of blood vessels and nerve fibers on the wound surface, so that the reconstruction of a neurovascular network is accelerated. In a word, the zinc silicate nanoparticle composite fiber scaffold can effectively promote wound healing and has the capability of reconstructing neurovascular network.
Claims (10)
1. A zinc silicate nanoparticle composite fibrous scaffold for simultaneously promoting healing of a skin wound and reconstruction of a cutaneous neurovascular network, comprising: the biological polymer fiber and the zinc silicate nano-particles embedded in the biological polymer fiber; the mass ratio of the zinc silicate nano particles to the biopolymer fibers is (0.02-0.4): 1;
the biological polymer fiber scaffold is a three-dimensional network structure formed by directionally arranged nano fibers;
the zinc silicate nano-particles are of a spindle-shaped structure formed by self-assembling and stacking zinc silicate nano-short rods, the length range is 600-900 nm, the width range is 200-500 nm, and the particle size distribution is 400-800 nm.
2. The zinc silicate nanoparticle composite fiber scaffold according to claim 1, wherein the biopolymer fibers are biocompatible polymer fibers, and the diameter of the fibers is in the range of 0.4-2 μm.
3. The zinc silicate nanoparticle composite fiber scaffold of claim 2, wherein the biopolymer fibers are polylactic acid, polycaprolactone or chitosan polymer fibers.
4. A method of preparing a zinc silicate nanoparticle composite fibrous scaffold according to any of claims 1 to 3, comprising: fully stirring and mixing a zinc salt solution, a silicon source solution and a sodium hydroxide solution to form a mixed solution, then carrying out hydrothermal reaction, collecting a precipitate and washing to obtain the zinc silicate nanoparticles; and uniformly mixing the zinc silicate nanoparticles and the biopolymer fiber material in a hexafluoroisopropanol solution, then filling the uniformly mixed solution into an injector for electrostatic spinning, and drying to obtain the zinc silicate nanoparticle composite fiber scaffold.
5. The preparation method according to claim 4, wherein the zinc salt is at least one of zinc nitrate, zinc nitrate hydrate, zinc sulfate hydrate, or anhydrous zinc chloride, and the silicon source is sodium silicate hydrate or tetraethoxysilane TEOS; the mass concentration of the zinc salt in the mixed solution is 0.05-0.1 g/mL, the mass concentration of the sodium hydroxide is 0.2-0.4 g/mL, and the mass concentration of the silicon source is 0.01-0.1 g/mL.
6. The preparation method according to claim 4, wherein the temperature of the hydrothermal reaction is 160-240 ℃ and the time is 12-36 h.
7. The preparation method according to claim 4, wherein the mass ratio of the zinc silicate nanoparticles to the biopolymer fiber material in the hexafluoroisopropanol solution is (0.02-0.4): 1.
8. The preparation method according to claim 7, wherein the mass ratio of the zinc silicate nanoparticles to the biopolymer fiber material in the hexafluoroisopropanol solution is 0.1.
9. The method according to claim 4, wherein the parameters of the electrospinning are: the spinning voltage is 8-12 kV, the rotation speed of the receiving roller is 1800-2500 r/min, the extrusion speed of the needle is 0.01-0.05 mL/min, and the distance between the receiving roller and the needle is 10-20 cm.
10. The use of the zinc silicate nanoparticle composite fibrous scaffold of claim 1 in a bioactive skin tissue engineering scaffold material with dual functions of wound healing and neurovascular network reconstruction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111505370.7A CN114272443B (en) | 2021-12-10 | 2021-12-10 | Preparation method and application of zinc silicate nanoparticle composite fiber scaffold |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111505370.7A CN114272443B (en) | 2021-12-10 | 2021-12-10 | Preparation method and application of zinc silicate nanoparticle composite fiber scaffold |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114272443A CN114272443A (en) | 2022-04-05 |
| CN114272443B true CN114272443B (en) | 2022-11-11 |
Family
ID=80871588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111505370.7A Active CN114272443B (en) | 2021-12-10 | 2021-12-10 | Preparation method and application of zinc silicate nanoparticle composite fiber scaffold |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114272443B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115137867B (en) * | 2022-04-26 | 2023-06-27 | 温州爱恩思生物科技有限公司 | Anti-scar material containing silicon-based inorganic material, preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101070162A (en) * | 2006-05-12 | 2007-11-14 | 中国科学院合肥物质科学研究院 | Composite hollow ball formed from radial densed-arranged zinc silicate nano wire and preparing method |
| CN102164615A (en) * | 2008-08-14 | 2011-08-24 | 索尼公司 | Drug sustained release agent, adsorbent, functional food, mask, and adsorptive sheet |
| CN105829486A (en) * | 2013-12-24 | 2016-08-03 | 东亚合成株式会社 | Aldehyde-gas-adsorbing liquid and gas-adsorbing processed product using same |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101856510B (en) * | 2010-05-14 | 2012-11-21 | 浙江理工大学 | Preparation method of composite nano-fiber support material of silk fibroin and calcium silicate |
| US20140050765A1 (en) * | 2012-08-14 | 2014-02-20 | Bio2 Technologies, Inc. | Devices and Methods for Tissue Engineering |
| SE1651271A1 (en) * | 2016-09-27 | 2018-03-28 | Gpbio Ltd | Composition of alfa-tcp, silicate and phosphorylated amino acid |
| CN109125782A (en) * | 2018-07-25 | 2019-01-04 | 华南理工大学 | A kind of porous fibre/inorganic bio Particles dispersed type skin wound dressing and preparation method thereof |
| CN112755241A (en) * | 2020-12-31 | 2021-05-07 | 无锡中科光远生物材料有限公司 | Dimethyloxalyl glycine-nano silicate fiber membrane and preparation method thereof |
| CN113499481B (en) * | 2021-06-18 | 2022-07-19 | 广东省科学院健康医学研究所 | Porous scaffold and preparation method and application thereof |
-
2021
- 2021-12-10 CN CN202111505370.7A patent/CN114272443B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101070162A (en) * | 2006-05-12 | 2007-11-14 | 中国科学院合肥物质科学研究院 | Composite hollow ball formed from radial densed-arranged zinc silicate nano wire and preparing method |
| CN102164615A (en) * | 2008-08-14 | 2011-08-24 | 索尼公司 | Drug sustained release agent, adsorbent, functional food, mask, and adsorptive sheet |
| CN105829486A (en) * | 2013-12-24 | 2016-08-03 | 东亚合成株式会社 | Aldehyde-gas-adsorbing liquid and gas-adsorbing processed product using same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114272443A (en) | 2022-04-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2460697C2 (en) | Silica sol-based material for producing biologically degradable and/or resorbable silica gel materials, production and use thereof | |
| Fu et al. | Skin tissue repair materials from bacterial cellulose by a multilayer fermentation method | |
| He et al. | Harnessing biocompatible nanofibers and silver nanoparticles for wound healing: Sandwich wound dressing versus commercial silver sulfadiazine dressing | |
| Cui et al. | Collagen-tussah silk fibroin hybrid scaffolds loaded with bone mesenchymal stem cells promote skin wound repair in rats | |
| Maraldi et al. | Human amniotic fluid stem cells seeded in fibroin scaffold produce in vivo mineralized matrix | |
| Biazar et al. | The healing effect of stem cells loaded in nanofibrous scaffolds on full thickness skin defects | |
| CN107233611A (en) | A kind of multifunctional nano fiber wound repair biological dressing and preparation method thereof | |
| Fu et al. | P34HB electrospun fibres promote bone regeneration in vivo | |
| Mao et al. | Construction of a multifunctional 3D nanofiber aerogel loaded with ZnO for wound healing | |
| CN112870439A (en) | Nano fiber bone tissue engineering scaffold with core-shell-series crystal structure and preparation method thereof | |
| CN110755408B (en) | Chitosan antibacterial microspheres capable of slowly releasing growth factors and preparation method and application thereof | |
| CN113318274A (en) | Hydrogel and preparation method and application thereof | |
| Xu et al. | 3D polycaprolactone/gelatin-oriented electrospun scaffolds promote periodontal regeneration | |
| CN114272443B (en) | Preparation method and application of zinc silicate nanoparticle composite fiber scaffold | |
| CN112972760A (en) | Endothelial extracellular matrix-loaded 3D printing bone defect repair stent and preparation method thereof | |
| CN110565207B (en) | Fiber material and preparation method and application thereof | |
| Yang et al. | Coaxial bioelectrospinning of P34HB/PVA microfibers biomimetic scaffolds with simultaneity cell-laden for improving bone regeneration | |
| Bakhtiary et al. | Wet-electrospinning of nanofibrous magnetic composite 3-D scaffolds for enhanced stem cells neural differentiation | |
| CN114316162B (en) | Photo-crosslinking injectable nanofiber-hydrogel compound as well as preparation method and application thereof | |
| CN108261557B (en) | Nanofiber membrane for wound healing and preparation method and application thereof | |
| Ma et al. | 3D printing of diatomite incorporated composite scaffolds for skin repair of deep burn wounds | |
| CN111744049A (en) | Preparation method of wound repair material with cell growth regulation function | |
| Wang et al. | Fabrication of an exosome-loaded thermosensitive chitin-based hydrogel for dental pulp regeneration | |
| WO2023179544A1 (en) | 3d-printed bone defect repair scaffold loaded with mesenchymal stem cell extracellular matrixes and preparation method therefor | |
| CN108079378B (en) | Organic/inorganic composite myocardial patch material with active ion release function and nano structure and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |