CN116003827A - Injectable hydrogel for promoting bone chitosan to complex zinc as well as preparation method and application thereof - Google Patents
Injectable hydrogel for promoting bone chitosan to complex zinc as well as preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the field of biological materials, and particularly relates to a preparation method and application of a low-zinc-content bone chitosan zinc complex injectable hydrogel. The injectable hydrogels include scheme 1 and scheme 2; the scheme 1 is that chitosan zinc hydrogel is directly used, and the proportion is as follows: the chitosan zinc accounts for 0.01 to 0.016 percent of the total mass of the hydrogel product, and the zinc element accounts for 0.0005 to 0.0008 percent of the total mass of the hydrogel product; the scheme 2 is as follows; firstly synthesizing high-concentration chitosan zinc hydrogel, and then diluting the chitosan zinc hydrogel and the diluent until zinc element accounts for 0.0005% -0.0008% of the total mass of the injectable hydrogel. The preparation method comprises the following steps: the chitosan zinc complex is prepared and then is prepared into hydrogel with a certain concentration. The product designed and prepared by the invention can be used as a human implantation material. The system has reasonable design, simple and controllable preparation process flow and strong efficacy; is suitable for large-scale application.
Description
Technical Field
The invention belongs to the field of biological materials, and particularly relates to a preparation method and application of injectable hydrogel for promoting chitosan to complex zinc.
Background
With the aggravation of aging population, the prevalence rate of osteoporosis and the prevalence rate of brittle fracture in China are increasing year by year. According to statistics, the osteoporosis patients in China in 2020 are up to 9000 ten thousand, accounting for 7.1% of the total population, the osteoporosis prevalence rate of female population over 50 years old is up to 29.1%, and the osteoporosis prevalence rate of male is 6.5%. It is estimated that by 2050, china will experience 544-655 thousands of fractures each year, with medical costs of about 254.3 billion dollars. Osteoporosis is a systemic bone metabolic disease characterized by low bone mass and destruction of bone tissue microstructure, resulting in increased bone fragility and hence fracture. In addition to fractures, osteoporosis can cause or exacerbate cardiovascular and cerebrovascular complications, lead to osteonecrosis and low healing rates, severe and even teratogenic disability. Therefore, how to effectively treat the fracture caused by osteoporosis and avoid the occurrence of infection in treatment at the same time becomes a focus of basic medicine of the current orthopedic treatment and a research hot spot of clinical application. The current widely used clinical schemes for treating bone defects include metal device fixation, bone autograft and allograft. However, most bioinert metals need to be removed by surgery, autografts may have a risk of difficult healing, and allografts may cause disease transmission and rejection.
Recently, bone tissue engineering has shown great promise in treating bone defects caused by osteoporosis. Bone tissue is a natural nanocomposite material consisting of organic proteins (type I collagen, COL as the main component), inorganic minerals (consisting of calcium phosphate) and various cells. Wherein bone is composed of a variety of characteristic cells surrounded by extracellular matrix (ECM), and thus bone ECM resembles a bone tissue engineering scaffold. The bone tissue engineering is that osteoblast, bone marrow stromal cell or chondrocyte are planted on degradable cell bracket after in vitro culture proliferation, then planted on bone defect part, the bracket is degraded and the bone cell proliferation process is carried out, finally the aim of repairing bone defect is achieved. Currently, common stent materials are metals, polymers, ceramics, and the like. The degradable metal implant is implanted into the body, the degradation time is as long as two and three years (more than 3-6 months of bone healing time), and the nondegradable metal material needs to be taken out for a second time, so that the medical cost is greatly increased.
Because of its characteristics of promoting wound healing and inhibiting bacteria, chitosan zinc has been widely studied, for example Hu Rui describes that carboxymethyl chitosan zinc can promote wound healing in its ' research on the effect of carboxymethyl chitosan zinc on promoting wound healing ' in its ' Shuoshsu. Wu Huiqing in the "antibacterial Property study of Chitosan and its Metal Zinc coordination Complex" it is described that Chitosan has excellent antibacterial Property. Meanwhile, some researches are also carried out on the osteogenesis performance, for example Qin Boheng, in the research on the osteogenesis performance of zinc-loaded multi-wall carbon nano tube/chitosan composite materials in the university paper of Qin Boheng, the osteogenesis performance of zinc-loaded multi-wall carbon nano tube/chitosan composite materials with different zinc loading amounts is introduced. In this paper, it was found that 0.2wt% zinc has limited ability to promote osteogenesis under this system.
Meanwhile, it has been found through searching that there are few zinc oligosaccharide complex zinc injectable hydrogels and reports of the zinc oligosaccharide complex zinc injectable hydrogels for osteogenesis.
Disclosure of Invention
In view of the shortcomings of the prior art, a first object of the present invention is to provide a chitosan zinc complex injectable hydrogel having both antibacterial and bone regeneration promoting effects over a long period of time.
The second aim of the invention is to provide a preparation method of the bone chitosan zinc complex injectable hydrogel, which is low in cost and high in efficiency.
A third object of the present invention is to provide the use of the chitosan zinc complex injectable hydrogel material.
The invention relates to an injectable hydrogel for promoting chitosan to complex zinc; the raw materials comprise zinc element, chitosan and sodium alginate, and the raw materials are in a hydrogel shape after reaction;
the injectable hydrogel includes scheme 1, scheme 2;
the scheme 1 is that chitosan zinc hydrogel is directly used, and the proportion is as follows: the chitosan zinc accounts for 0.01 to 0.016 percent of the total mass of the hydrogel product, and the zinc element accounts for 0.0005 to 0.0008 percent of the total mass of the hydrogel product;
the scheme 2 is as follows: firstly synthesizing high-concentration chitosan zinc hydrogel, and then diluting the chitosan zinc hydrogel and the diluent until zinc element accounts for 0.0005% -0.0008% of the total mass of the injectable hydrogel. The invention realizes the excellent bone regeneration effect of chitosan zinc with low zinc content.
The dilution liquid comprises a cell culture liquid and/or water. The cell culture fluid comprises a class A culture fluid; the A-type culture solution consists of 89% of a-MEM+10% of serum+1% of diabody by volume percentage. The diabody is typically a mixture of green streptomycin in the art.
The invention relates to an injectable hydrogel for promoting chitosan to complex zinc; the zinc element and chitosan are subjected to complexation to obtain chitosan zinc complex.
The invention relates to an injectable hydrogel for promoting chitosan to complex zinc; the chitosan zinc complex is coated in the hydrogel, the hydrogel is porous, the pore diameter is 75-550 mu m, and the pore diameter is preferably 80-175 mu m.
The zinc ions have double slow release effects due to complexation of chitosan complexing zinc and hydrogel coating, and the zinc ions are in PBS solution at 37 ℃; on day 14, the release amount of zinc ions in zinc acetate is 2.4+/-0.1 mg/L, the release amount of zinc ions after chitosan complexing zinc is 0.6+/-0.08 mg/L, and the release amount of zinc ions in the hydrogel is further reduced to 0.3+/-0.05 mg/L (the high-concentration hydrogel obtained in scheme 2) after chitosan complexing zinc is added, so that the hydrogel has excellent antibacterial property on staphylococcus aureus; the hydrogels have good cell proliferation and differentiation capabilities and the products are prepared using chemical synthesis.
Namely: in PBS solution at 37deg.C; the release amount of zinc ions in the bone chitosan complex zinc injectable hydrogel obtained by the invention is 0.3+/-0.05 mg/L.
The invention relates to a preparation method of injectable hydrogel for promoting chitosan to complex zinc; after conversion according to the designed components, mixing zinc acetate ethanol solution and chitosan for reaction, and centrifugally drying to obtain the chitosan zinc complex; and mixing the obtained chitosan zinc complex with a sodium dodecyl sulfate aqueous solution, respectively adding sodium alginate, acrylamide, octadecyl methacrylate, potassium persulfate and tetramethyl ethylenediamine, and drying to obtain the product.
As an optimal scheme, the preparation method of the injectable zinc hydrogel for promoting the bone chitosan to complex zinc has the advantages that chitosan is of a lamellar structure of 200-300 mu m, and the deacetylation degree is 90%.
As a preferred scheme, the preparation method of the bone chitosan zinc complex injectable hydrogel is porous, the pore diameter of the hydrogel is 65-550 mu m, preferably 70-170 mu m, and further preferably 80-170 mu m. Wherein, the aperture of the compact bone is about 100 mu m, and the preferred aperture of the rear hydrogel is 80-170 mu m, which is more fit with the requirement of bone formation on the surface structure, is favorable for cell attachment and pseudopodia growth, and promotes the subsequent bone regeneration process. During the development of the invention, a method for regulating the pore diameter of the hydrogel is discovered.
In the discovery process of the present invention, the hydrogel pore size was found to be slightly smaller than the pore size of the product obtained in scheme 2 when prepared in scheme 1. In industrial application, the chitosan complex zinc content is slightly higher than the zinc concentration defined in the scheme 1, the pore diameter is controlled by the chitosan complex zinc content, and finally, after the gel is prepared, water or cell culture solution is replenished again to form a target product.
As a further preferable scheme, the preparation method of the injectable zinc complex bone chitosan hydrogel comprises the steps of preparing ethanol solution by preparing deionized water and absolute ethanol in a volume of 1:18-20, preferably 1:19, and dissolving zinc acetate in the ethanol solution to prepare zinc acetate ethanol solution; at a concentration of 1 g/L; adding chitosan into the zinc acetate ethanol solution, sealing with tinfoil, placing into a heat-collecting constant-temperature heating magnetic stirrer, and continuously stirring in a water bath at 55-65deg.C for 3-5 hr, preferably 4 hr for reaction. After the reaction is finished, the solution is split into centrifuge tubes, centrifuged for 4-6min at 8000-12000rpm, and the obtained precipitate is washed with absolute ethyl alcohol, and the washing is repeated for at least 2 times. And (3) placing the washed precipitate in a vacuum drying oven, and drying at 55-65 ℃ for 20-28h, wherein the chitosan is complexed with zinc. Adding sodium dodecyl sulfate and chitosan zinc complex into deionized water to form an aqueous solution containing chitosan zinc complex, and magnetically stirring until the aqueous solution is uniform. Adding sodium alginate and acrylamide into the aqueous solution containing chitosan zinc complex for crosslinking, and continuously stirring for 3-5h, preferably 4h; after that, octadecyl methacrylate is added and magnetically stirred for 8 to 12 hours, preferably 10 hours. After adding potassium persulfate, stirring is continued for 25-35min to uniformly distribute the potassium persulfate. Then adding tetramethyl ethylenediamine, stirring for 15-25min, pouring into a mold, standing and drying.
Preferably, 0.4-0.6g of sodium dodecyl sulfate and 80-150mg of chitosan zinc complex are added into 95-105mL of deionized water according to a proportion to form an aqueous solution containing chitosan zinc complex, and magnetic stirring is carried out until the aqueous solution is uniform; according to the proportion, 100mL of aqueous solution containing chitosan complex zinc is added with 2.0-4.0g of sodium alginate, 0.5-1.5g of acrylamide, 0.5-1mL of octadecyl methacrylate and 0.03-0.09g of potassium persulfate, and sodium alginate and acrylamide are added into the obtained aqueous solution containing chitosan complex zinc for crosslinking, and stirring is continued for 3-5 hours, preferably 4 hours; adding octadecyl methacrylate, and magnetically stirring for 8-12h, preferably 10h; after adding potassium persulfate, stirring is continued for 25-35min to uniformly distribute the potassium persulfate. Then adding tetramethyl ethylenediamine, stirring for 15-25min, pouring into a mold, standing and drying. At this time, a high concentration chitosan zinc hydrogel was obtained.
As a further preferable scheme, the chitosan zinc complex has a lamellar structure of 200-300 mu m.
In the invention, the proportion is calculated; deionized water and absolute ethanol were prepared in a volume of 1:19 to prepare 200mL of ethanol solution, and 200mg of zinc acetate was weighed and dissolved therein to prepare 1g/L of zinc acetate ethanol solution (95% ethanol). Weighing 4g of chitosan, adding the chitosan into the zinc acetate ethanol solution, sealing the tin foil, placing the tin foil into a heat-collecting constant-temperature heating magnetic stirrer, and continuously stirring in a water bath at 60 ℃ for 4 hours to perform a reaction. After the reaction was completed, the solution was dispensed into 50mL centrifuge tubes, centrifuged at 10000rpm for 5min, and the obtained precipitate was washed with absolute ethanol, and washing was repeated 2 times. And (3) placing the washed precipitate in a vacuum drying oven and drying at 60 ℃ for 24 hours to obtain a yellow chitosan zinc complex product with zinc content of 5 wt%.
The invention relates to an application of an injectable zinc complex chitosan hydrogel, which comprises the application of the injectable zinc complex chitosan hydrogel serving as a human implantation material.
Drawings
FIG. 1 shows the macroscopic and microscopic morphology of the product obtained in example 1 and of chitosan;
FIG. 2 is an elemental distribution diagram of the product obtained in example 1 and chitosan;
FIG. 3 is an XRD characterization and UV pattern of the product obtained in example 1;
FIG. 4 is a graph of the macro morphology of the product SA/PAAM-300 obtained in example 2;
FIG. 5 is a microstructure of the product of example 2;
FIG. 6 is a graph showing the cumulative zinc ion release of the products obtained in examples 1 and 2 and zinc acetate;
FIG. 7 is a graph showing the bacteriostatic effects of the products of example 2 and comparative example 4 on Staphylococcus aureus.
FIG. 8 shows the cytoskeletal morphology of the products obtained in example 3 and comparative example 5.
FIG. 9 is a graph showing the results of qualitative and quantitative characterization of alkaline phosphatase activity in example 3.
FIG. 10 is a graph showing the results of quantitative characterization of ALP activity on the fifth day of the blank group and the experimental group.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings.
Example 1
Deionized water and absolute ethanol were prepared in a volume of 1:19 to prepare 200mL of ethanol solution, and 200mg of zinc acetate was weighed and dissolved therein to prepare 1g/L of zinc acetate ethanol solution (95% ethanol). Weighing 4g of chitosan, adding the chitosan into the zinc acetate ethanol solution, sealing the tin foil, placing the tin foil into a heat-collecting constant-temperature heating magnetic stirrer, and continuously stirring in a water bath at 60 ℃ for 4 hours to perform a reaction. After the reaction was completed, the solution was dispensed into 50mL centrifuge tubes, centrifuged at 10000rpm for 5min, and the obtained precipitate was washed with absolute ethanol, and washing was repeated 2 times. And (3) placing the washed precipitate in a vacuum drying oven and drying at 60 ℃ for 24 hours to obtain a yellow chitosan zinc complex product with zinc content of 5 wt%.
Effect of the invention
Comparing the macro morphology of CS-Zn with the macro morphology of chitosan (the macro graph is shown as figure 1 (a)) and the macro morphology of CS-Zn (the macro graph is shown as figure 1 (c)), the chitosan has the obvious change in color from white to yellow compared with the chitosan. The microscopic morphology of the chitosan is shown in fig. 1 (b), the microscopic morphology of the CS-Zn is shown in fig. 1 (d) and the microscopic morphology of the CS-Zn is shown in fig. 1 (b) and fig. 1 (d), and the CS-Zn is shown as a lamellar structure of 200-300 μm without obviously changing the shape and the size of the CS-Zn compared with the chitosan. As shown in FIG. 2 (a), the CS-Zn element distribution is that Zn element is contained in the sample, and as shown in FIG. 2 (b), zn element is uniformly distributed therein. The XRD patterns of chitosan in this patent had two distinct diffraction peaks of 11.76℃and 20.08℃as shown in FIG. 3 (a). The XRD pattern of CS-Zn showed a distinct decrease in both diffraction peaks, especially at 11.76 deg.. It can be seen that the crystallinity of chitosan is reduced due to the addition of zinc acetate. Further analysis FIG. 3 (b), chitosan shows absorption at 201.5nmThe absorption peak of CS-Zn is at 202.0nm, namely the absorption peak of the product is red shifted. From this, it was found that chemical bonding of chitosan and zinc acetate occurred, resulting in n-sigma existing in chitosan * The transition is affected resulting in a red shift of the absorption peak. Thus, zinc ions in zinc acetate are successfully complexed into chitosan.
0.5g of sodium dodecyl sulfate and 100mg of chitosan or 100-300mg of CS-Zn are respectively added into 100mL of deionized water to form CS aqueous solution or CS-Zn-containing aqueous solution with mass fraction of 0.098% -0.29%, and magnetic stirring is carried out for 30 min. Adding 2.5g of sodium alginate into the solution at the same time, then adding 1.5g of acrylamide for crosslinking, and continuously stirring for 4 hours; 780. Mu.L of octadecyl methacrylate was added and magnetically stirred for 10h. After adding 0.06g of potassium persulfate, stirring was continued for 30 minutes to uniformly distribute it. Subsequently, 120 mu L of tetramethyl ethylenediamine is added, and after stirring for 20min, 1mL of the mixture is injected into a 24-well plate, and finally, the CS-Zn hydrogel with the Zn content of 0.005% -0.283% is formed, namely, the CS-Zn hydrogel has the mass fraction of 0.098% -0.29% in the hydrogel and the chitosan hydrogel has the mass fraction of 0.098 wt%.
Depending on the amount of CS and CS-Zn used in the prepared hydrogels, 100mg CS was added as SA/PAAM-0, and 100mg, 200mg and 300mg CS-Zn were added as SA/PAAM-100 (the Zn content in the hydrogels was 0.005wt%, the chitosan zinc complex content was 0.098 wt%), SA/PAAM-200 (the Zn content in the hydrogels was 0.01wt%, the chitosan zinc complex content was 0.20 wt%) and SA/PAAM-300 (the Zn content in the hydrogels was 0.015wt%, the chitosan zinc complex content was 0.29 wt%).
Effect of the invention
FIG. 4 shows the macroscopic morphology of a hydrogel, with half flowability observed at the beginning of synthesis; after being placed in a constant temperature box at 37.5 ℃ for 4 hours and cured and molded, the hydrogel can be observed to shrink slightly in volume after the curing and molding. The internal morphology and elements of the hydrogels are shown in fig. 5, and a distinct three-dimensional network structure can be observed for each group of samples. As can be seen from FIG. 5 (a), wherein the pore size of SA/PAAM-0 is about 62-160. Mu.m, from FIG. 5 (b), SA/PAAM-100 is about 80-170. Mu.m, from FIG. 5 (c), SA/PAAM-200 is about 110-250. Mu.m, and from FIG. 5 (d), SA/PAAM-300 is about 230-550. Mu.m, the pore size of the hydrogel tends to increase as the CS-Zn content in the hydrogel increases. Wherein the dense bone has a pore size of about 100 μm, and thus has an average pore size of about 100 μm for both SA/PAAM-0 and SA/PAAM-100 samples. The surface structure is more fit with the osteogenesis requirement, which is beneficial to cell attachment and pseudopodia growth and promotes the subsequent bone regeneration process. Meanwhile, in the process of the series of experimental exploration, some measures for controlling the pore diameter of the hydrogel are also discovered, such as adjusting the dosage of Cs-Zn to be matched with other raw materials.
To prove CS-Zn vs. Zn 2+ Has slow release effect, and can be used as medicine to be added into hydrogel for Zn 2+ Release behavior, respectively soaking 100mg zinc acetate, 100mg CS-Zn and SA/PAAM-100 hydrogel in 10ml PBS solution at 25deg.C for 14 days, and Zn 2+ The result of the cumulative release amount of (2) is shown in fig. 6. Zinc acetate releases Zn up to 1.57mg/L on the first day 2+ Up to day seven reaching 2.36mg/L, it releases Zn 2+ Far greater than CS-Zn and hydrogels. CS-Zn releases relatively rapidly in the first four days, zn 2+ The concentration rapidly reached 0.49mg/L. However, the release rate gradually becomes slow with the prolonged soaking time, and Zn is released from day 10 to day 14 2+ The release concentration is stabilized at 0.64mg/L; whereas the hydrogel rapidly released Zn on the first day 2+ The concentration reaches 0.08mg/L; the release rate gradually becomes gentle after the first day, zn 2+ The concentration was slowly raised to 0.17mg/L. But Zn in seventh to tenth days 2+ The release rate was increased again and increased to 0.30mg/L on the tenth day; the release rate gradually becomes gentle again from the tenth day to the fourteenth day, and gradually stabilizes at 0.31mg/L. In general, zinc acetate is complexed into CS-Zn, zn 2+ Release amount of Zn 2+ A decrease in release rate. After CS-Zn is added into the hydrogel, zn 2+ The release rate was further reduced and there was a pronounced slow release behavior. The CS-Zn is considered to be a mesh structure of the hydrogel coated in the reaction, and the unique three-dimensional network structure in the hydrogel has obvious slow release effect on the CS-Zn. Hydrogel-likeThe product shows a certain antibacterial property to staphylococcus aureus, as shown in figure 7, the periphery of SA/PAAM-0 has no antibacterial zone, the antibacterial zone of SA/PAAM-100 is maximum, the antibacterial effect is optimal, however, the antibacterial effects of SA/PAAM-200 and SA/PAAM-300 with increased drug loading are not obviously improved. Thus, the SA/PAAM-100 group has reached the antimicrobial threshold. The SA/PAAM-100 samples are preferred for subsequent cell experiments, since they bind to surface topography.
Example series 3
According to national standard GB/T16886.5-2017, the hydrogel sample SA/PAAM-100 is prepared according to 1.25cm 2 Standard per mL, a-MEM medium was used as the leaching medium, the uv sterilized hydrogel was placed in a cell incubator for 72h and filtered with a sterile needle filter (r=0.22 μm) after the end of leaching to prepare a complete leaching solution of SA/PAAM-100 (approximately believing that Zn in SA/PAAM-100 had been completely leached).
According to national standard GB/T16886.5-2017, the hydrogel sample SA/PAAM-0 is prepared according to 1.25cm 2 Standard per mL, a-MEM medium was used as the leaching medium, the uv sterilized hydrogel was placed in a cell incubator for 72h and filtered with a sterile needle filter (r=0.22 μm) after the leaching was completed to prepare a complete leaching solution of SA/PAAM-0.
Setting a blank group:
preparing a cell culture solution (89% a-MEM+10% serum+1% double antibody) and marking the cell culture solution as a blank control group;
setting an experimental group:
taking SA/PAAM-0 leaching solution with the volume of V, diluting with 9V cell culture solution (89% a-MEM+10% serum+1% double antibody) to obtain SA/PAAM-0 leaching solution diluted to 10%, and calculating as CS-II;
taking SA/PAAM-100 leaching solution with volume of V, diluting with 19V cell culture solution (89% a-MEM+10% serum+1% double antibody) to obtain SA/PAAM-100 leaching solution diluted to 5%, and calculating as Zn-I;
taking SA/PAAM-100 leaching solution with the volume of V, diluting with 9V cell culture solution (89% a-MEM+10% serum+1% double antibody) to obtain SA/PAAM-100 leaching solution diluted to 10%, and counting as Zn-II;
taking SA/PAAM-100 leaching solution with the volume of V, diluting with 5.65V cell culture solution (89% a-MEM+10% serum+1% double antibody) to obtain SA/PAAM-100 leaching solution diluted to 15%, wherein the leaching solution is calculated as Zn-III;
effect of the invention
In cytotoxicity tests on day 1, day 3 and day 5 of MC3T3-E1 cells, it can be seen that Zn-II group had better bioactivity (Table 1).
TABLE 1 OD values of cells at 450nm wavelength in each set of samples
The cytoskeleton of MC3T3-E1 co-cultured with the sample for 24h is shown in FIG. 8. Wherein blue is nucleus and green is cytoplasm. FIG. 8 (a) is a cytoskeletal profile of MC3TS-E1 cells in a blank incubated for 24h on a 48-well plate; FIG. 8 (b) is a cytoskeletal profile of MC3TS-E1 cells in CS-II groups incubated for 24h on 48-well plates; FIG. 8 (d) is a cytoskeletal profile of MC3TS-E1 cells in group Zn-I incubated for 24h on 48-well plates; FIG. 8 (E) is a cytoskeletal profile of MC3TS-E1 cells in group Zn-III incubated for 24h on 48 well plates; fig. 8 (f) is an enlarged view of the selected area in fig. 8 (c). As can be seen from the graph, the number of cells in each experimental group is equivalent to that in the blank control group, the morphology is not obviously different, and the cell attachment and the cell spreading are good.
Alkaline phosphatase (ALP) activity is an early signal of osteogenic differentiation. Continuously culturing the blank control group, the CS-II, the Zn-I, the Zn-II and the Zn-III group until the fifth day, and performing qualitative and quantitative treatment by using an ALP kit to obtain a result shown in a graph 9, wherein (a) of the graph 9 is an ALP activity qualitative characterization result of MC3TS-E1 cells in the blank control group incubated on a 48-pore plate for 5 days; FIG. 9 (b) is a qualitative characterization of ALP activity in MC3TS-E1 cells from CS-II group incubated for 5 days on 48-well plates; FIG. 9 (d) is a qualitative characterization of ALP activity in MC3TS-E1 cells in Zn-I groups incubated for 5 days on 48-well plates; FIG. 9 (E) is a qualitative characterization of ALP activity in MC3TS-E1 cells in Zn-III groups incubated for 5 days on 48-well plates. CS-II, zn-I and Zn-II all promoted ALP activity compared to the placebo group, wherein Zn-II significantly promoted ALP activity. And comparing the three groups Zn-I, zn-II and Zn-III, the ALP activity is promoted and inhibited firstly with the increase of the concentration of the leaching solution. Thus, zn-II exhibits optimal early osteogenic differentiation behavior. Based on the above, it is considered that the chitosan zinc accounts for 0.01 to 0.016 percent of the total mass of the hydrogel product, and the chitosan-containing zinc complex injectable hydrogel with the Zn content of 0.0005 to 0.0008 percent by weight is beneficial to bone formation in an implant body.
Comparative example 1
Deionized water and absolute ethanol were prepared in a volume of 1:19 to prepare 200mL of ethanol solution, and 40mg of zinc acetate was weighed and dissolved therein to prepare 0.2g/L of zinc acetate ethanol solution (95% ethanol). Adding the zinc acetate ethanol solution into 4g of chitosan, sealing with tinfoil, placing into a heat-collecting constant-temperature heating magnetic stirrer, and continuously stirring in a water bath at 60 ℃ for 4 hours to react, so as to prepare the medicament with zinc content of 1%. After the reaction was completed, the solution was dispensed into 50mL centrifuge tubes, centrifuged at 10000rpm for 5min, and the obtained precipitate was washed with absolute ethanol, and washing was repeated 2 times. And (3) drying the washed precipitate in a vacuum drying oven at 60 ℃ for 24 hours, wherein zinc acetate is dissolved in a solvent and does not react with chitosan chemically, so that a white chitosan zinc uncomplexed product is obtained.
Comparative example 2
Deionized water and absolute ethanol were prepared in a volume of 1:19 to prepare 200mL of ethanol solution, and 400mg of zinc acetate was weighed and dissolved therein to prepare 2g/L of zinc acetate ethanol solution (95% ethanol). Adding the zinc acetate ethanol solution into 4g of chitosan, sealing with tinfoil, placing into a heat-collecting constant-temperature heating magnetic stirrer, and continuously stirring in a water bath at 60 ℃ for 4h to perform a reaction. After the reaction was completed, the solution was dispensed into 50mL centrifuge tubes, centrifuged at 10000rpm for 5min, and the obtained precipitate was washed with absolute ethanol, and washing was repeated 2 times. And (3) drying the washed precipitate in a vacuum drying oven at 60 ℃ for 24 hours, wherein zinc acetate is dissolved in a solvent and does not react with chitosan chemically, so that a white chitosan zinc uncomplexed product is obtained.
Comparative example 3
To 100mL of deionized water, 0.5g of sodium dodecyl sulfate was added, and the mixture was magnetically stirred for 30 minutes. 2.5g of sodium alginate and 1.5g of acrylamide are added into the solution at the same time, and stirring is continued for 4 hours; 780. Mu.L of octadecyl methacrylate was then added and magnetically stirred for 10h. After adding 0.06g of potassium persulfate, stirring was continued for 30 minutes to uniformly distribute it. Then 120 mu L of tetramethyl ethylenediamine is added, finally chitosan zinc complex is added, after stirring for 20min, 1mL of the mixture is injected into a 24-pore plate per pore, and the porous hydrogel structure is not obtained after surface morphology observation.
Comparative example 4
The different hydrogel samples (SA/PAAM-0, SA/PAAM-200 and SA/PAAM-300) all showed a certain antibacterial property against staphylococcus aureus, as shown in FIG. 6, wherein the SA/PAAM-0 group has almost no antibacterial effect, and compared with the SA/PAAM-100 sample, the antibacterial effect of the SA/PAAM-200 and the SA/PAAM-300 with increased drug loading is not obviously improved.
Comparative example 5
Cell proliferation experiments are carried out by using 15% of SA/PAAM-100 carrier hydrogel leaching liquor, and the result shows that the excessive dosage of the medicine can cause cytotoxicity and inhibit cell proliferation, and simultaneously influence the growth of cell pseudopodia.
Claims (10)
1. An injectable hydrogel of zinc complex forcing bone chitosan; the method is characterized in that: the raw materials comprise zinc element, chitosan and sodium alginate, and the raw materials are in a hydrogel shape after reaction;
the injectable hydrogels include scheme 1 and scheme 2;
the scheme 1 is that chitosan zinc hydrogel is directly used, and the proportion is as follows: the chitosan zinc accounts for 0.01 to 0.016 percent of the total mass of the hydrogel product, and the zinc element accounts for 0.0005 to 0.0008 percent of the total mass of the hydrogel product;
the scheme 2 is as follows; firstly synthesizing high-concentration chitosan zinc hydrogel, and then diluting the chitosan zinc hydrogel and the diluent until zinc element accounts for 0.0005% -0.0008% of the total mass of the injectable hydrogel.
2. An injectable hydrogel of zinc contributing to the complexation of chitosan with bone according to claim 1; the method is characterized in that: the zinc element and chitosan are subjected to complexation to obtain chitosan zinc complex.
3. An injectable hydrogel of zinc contributing to the complexation of chitosan with bone according to claim 1; the method is characterized in that: the chitosan zinc complex is coated in the hydrogel, the hydrogel is porous, the pore diameter is 75-550 mu m, and the pore diameter is preferably 80-175 mu m.
4. An injectable hydrogel of zinc contributing to the complexation of chitosan with bone according to claim 1; the method is characterized in that: in PBS solution at 37 ℃, the release amount of zinc ions in the chitosan complex zinc injectable hydrogel at 14 days is 0.31+/-0.05 mg/L.
5. A method of preparing an injectable hydrogel of any one of claims 1-4 that facilitates chitosan complexing zinc; the method is characterized in that: the chitosan zinc complex is obtained by mixing zinc acetate ethanol solution and chitosan for reaction, and centrifugally drying; and mixing the obtained chitosan zinc complex with a sodium dodecyl sulfate aqueous solution, respectively adding sodium alginate, acrylamide, octadecyl methacrylate, potassium persulfate and tetramethyl ethylenediamine, and drying to obtain the product.
6. The method for preparing the injectable zinc complex bone chitosan hydrogel according to claim 5, wherein the method comprises the following steps: the chitosan has a lamellar structure of 200-300 μm, and the deacetylation degree is 90%.
7. The method for preparing the injectable zinc complex bone chitosan hydrogel according to claim 5, wherein the method comprises the following steps: preparing ethanol solution by preparing deionized water and absolute ethanol in a volume of 1:18-20, preferably 1:19, and dissolving zinc acetate in the ethanol solution to prepare zinc acetate ethanol solution; adding chitosan into the zinc acetate ethanol solution according to the concentration of 1+/-0.1 g/L, sealing the tin foil, placing the tin foil into a heat-collecting constant-temperature heating magnetic stirrer, and continuously stirring in a water bath at 55-65 ℃ for 3-5 hours, preferably 4 hours for reaction; after the reaction is finished, subpackaging the solution into a centrifuge tube, centrifuging for 4-6min at 8000-12000rpm, washing the obtained precipitate with absolute ethyl alcohol, and repeatedly washing for at least 2 times; placing the washed precipitate in a vacuum drying oven, and drying at 55-65deg.C for 20-28 hr to obtain chitosan zinc complex;
adding sodium dodecyl sulfate and chitosan zinc complex into deionized water to form an aqueous solution containing chitosan zinc complex, and magnetically stirring until the aqueous solution is uniform; adding sodium alginate and acrylamide into the aqueous solution containing chitosan zinc complex for crosslinking, and continuously stirring for 3-5h, preferably 4h; adding octadecyl methacrylate, and magnetically stirring for 8-12h, preferably 10h; after adding potassium persulfate, stirring is continued for 25-35min to uniformly distribute the potassium persulfate. Then adding tetramethyl ethylenediamine, stirring for 15-25min, pouring into a mold, standing and drying.
8. The method for preparing the injectable zinc complex bone chitosan hydrogel according to claim 7, wherein the method comprises the following steps: adding 0.4-0.6g of sodium dodecyl sulfate and 80-150mg of chitosan zinc complex into 95-105mL of deionized water according to a proportion to form an aqueous solution containing the chitosan zinc complex, and magnetically stirring until the aqueous solution is uniform; according to the proportion, 100mL of aqueous solution containing chitosan complex zinc is added with 2.0-4.0g of sodium alginate, 0.5-1.5g of acrylamide, 0.5-1mL of octadecyl methacrylate and 0.03-0.09g of potassium persulfate, and sodium alginate and acrylamide are added into the obtained aqueous solution containing chitosan complex zinc for crosslinking, and stirring is continued for 3-5 hours, preferably 4 hours; adding octadecyl methacrylate, and magnetically stirring for 8-12h, preferably 10h; adding potassium persulfate, and continuing stirring for 25-35min to uniformly distribute the potassium persulfate; then adding tetramethyl ethylenediamine, stirring for 15-25min, injecting into a mold, standing and drying; obtaining the high-concentration chitosan zinc hydrogel.
9. The method for preparing the injectable zinc complex bone chitosan hydrogel according to claim 5, wherein the method comprises the following steps: the chitosan zinc complex has a lamellar structure of 200-300 mu m; the obtained hydrogel is porous, and has a pore size of 75 μm-550 μm, preferably 80-175 μm.
10. Use of the injectable hydrogel of any one of claims 1-4 to facilitate complexing zinc with chitosan, characterized in that: the application comprises the application of the material as a human body implantation material.
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