Bioactivity, in the field of materials, mainly refers to the characteristics that can induce special biological and chemical reactions at the interface between materials and biological tissues, which lead to the formation of chemical bonds between materials and biological tissues.In the process of biomineralization, it mainly refers to the ability of biomaterials to generate chemical bonding with living bones, which is an important indicator of biomaterials. The biological activity of materials in vivo can be reflected by the ability of material surface to form apatite in simulated body fluid SBF,The application of this method to evaluate the biological activity of materials can reduce the number of animals needed for experiments and increase the duration of animal experiments
Chinese name
biological activity
Meaning
Induced on the interface between material and biological tissue
This concept was discovered and put forward by American L. Hench in 1969 when he studied bioglass, and then introduced the concept of bioactivity in the field of bioceramics, creating a new research field.After more than 30 years of development, the concept of bioactivity has established a solid foundation in the field of biomaterials, such as β - tricalcium phosphate absorbable bioceramics, which can be degraded and absorbed in the body and replaced by new tissues, and has the function of inducing special biological reactions;Hydroxyapatite is the main inorganic component of natural bone, so it can not only conduct osteogenesis, but also form bone bond with new bone when implanted in the body. When implanted in muscles, ligaments or subcutaneously, it can close to tissues without inflammation or irritation.Living biomaterials have these special biological properties, which are conducive to the repair of human tissues. It is an important direction of biomaterials research and development.
Common materials
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Calcium phosphate material
Calcium phosphate bioactive materials mainly include calcium phosphate cement and calcium phosphate ceramic fiber.The former is a new material widely used for bone repair and joint fixation, and the compressive strength of domestic research has reached more than 60MPa.The latter has a certain mechanical strength and biological activity, which can be used for reinforcing inorganic bone cement and preparing organic and inorganic composite implant materials.Calcium phosphate fiber or whisker has good biological activity and biocompatibility, and has no side effects on human body. It is an ideal reinforcing material for bioceramics and organic polymer materials.
hydroxyapatite
Hydroxyapatite is one of the most studied bioactive materials at present, as the most representative bioactive ceramic - hydroxyapatite [Ca10 (P04) 6 (OH) 2, referred to as HA].Hydroxyapatite has the same main inorganic components as vertebrate bones and teeth, and its structure is very similar. It has good compatibility with animal tissues, no side effects, and its interfacial activity is superior to various medical titanium alloys, silicone rubber, and carbon materials for bone graft.It can be widely used in the repair and replacement of biological hard tissues, such as oral implants, alveolar ridge augmentation, ear bone replacement, spine bone replacement and many other aspects.In addition, it is also widely used in HA bioceramic middle ear ventilation drainage tube, maxillofacial bone, nasal bridge, pseudoeyeball, HA particles for filling and HA microcrystalline powder for inhibiting cancer cells.Hydroxyapatite is limited by its high brittleness and low flexural strength, so its application in load-bearing materials is limited.At present, the preparation of porous ceramics and composites is an important development direction of this material, and the preparation of coating materials is also one of its important branches.
Bioactive glass
Bioactive glass is a kind of material that can repair, replace and regenerate body tissues and can form bonds between tissues and materials.Bioactive glass (BAG) was discovered by Hench in 1969. It is a silicate glass composed of SiO2, Na2O, CaO and P2O5.The degradation products of bioactive glass can promote the generation of growth factors, promote the proliferation of cells, enhance the gene expression of osteoblasts and the growth of bone tissue.So far, it is the only artificial biomaterial that can not only bond with bone tissue, but also connect with soft tissue.
Activity test
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Osteogenic activity
The ability of the material surface to form apatite in SBF can reflect the biological activity of the material in vivo. The specific detection methods are as follows:
1. SBF configuration
Because SBF solution is supersaturated with apatite, improper preparation method will lead to apatite precipitation in the solution.The whole preparation process needs to ensure that the solution is colorless and transparent, and there is no precipitation on the surface of the container. If precipitation occurs in the process, pour out the solution, clean the instrument and prepare again.Prepare a 1000ml plastic beaker.First, fill a plastic beaker with 700ml of ion exchange distilled water, a magneton of appropriate size, and seal the mouth of the beaker with an evaporating dish or plastic film.Stir and adjust the water temperature to 36.5 ± 1.5C.
Dissolve the first to the eighth compounds one by one under the constant temperature of 36.5C in the order listed in Table 1, and the dissolution process
Table 1 Reagent addition sequence and amount when preparing 1000 ml SBF solution
Order
Reagents
Amounts
Containers
Purities（%）
Formula weights
one
NaCl
eight point zero three five
Weight paper
ninety-nine point five
fifty-eight point four four three zero
two
NaHCO3
zero point three five five
Weight paper
ninety-nine point five
eighty-four point zero zero six eight
three
KCl
zero point two two five
Weight bottle
ninety-nine point five
seventy-four point five five one five
four
K2HPO4·3H2O
zero point two three one
Weight bottle
ninety-nine
two hundred and twenty-eight point two two two zero
five
MgCl2·6H2O
zero point three one one
Weight bottle
ninety-eight
two hundred and three point three zero three four
six
1.0 M-HCl
39ml
Graduated cylinder
—
—
seven
CaCl2
zero point two nine two
Weight bottle
ninety-five
one hundred and ten point nine eight four eight
eight
Na2SO4
zero point zero seven two
Weight bottle
ninety-nine
one hundred and forty-two point zero four two eight
nine
Tris
six point one one eight
Weight paper
ninety-nine
one hundred and twenty-one point one three five six
ten
1.0 M-HCl
0-5ml
Syringe
—
—
2. Apatite forming ability test
For dense sheet materials, measure the sample size and calculate the surface area of the sample to the nearest 2mm, and calculate the amount of SBF using the following formula:
Vs=Sa/10,
Vs (ml) is the volume of SBF, and Sa (mm) is the surface area of the sample.
For porous materials, the amount of SBF should be greater than the amount calculated in the above formula. Prepare a plastic bottle or beaker, fill it with the calculated SBF volume and heat it to 36.5C, and then place the sample according to the schematic diagram.Note: There are two cases when the sample is placed in the container, as shown in Figure 1 (a) and 1 (b).In a few cases, SBF solution will generate homogeneous apatite to deposit on the sample surface.Therefore, if the sample is placed as shown in Figure A1 (b), the characterization experiment should detect the lower surface of the sample.Within four weeks, take out the samples of each group that have been soaked at constant temperature for different periods of time, and clean them with pure water.Then put the sample into a dryer to dry at room temperature.
Figure 1 Schematic Diagram of Sample Soaking in SBF
Cellular activity
1. Tetrazolium salt (MTT) colorimetry:
In addition to the methods described above, tetrazolium salt colorimetry (MTT) can also be used to detect cell survival and growth.The principle of this method is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into insoluble blue purple crystals and deposit them in cells, but cells have no such function.Dimethyl sulfoxide (DMSO) can dissolve the blue purple crystal in cells, and use ELISA to determine its light absorption value at 490nm wavelength, which can indirectly reflect the number of living cells.Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells.
● The cells were digested by 0.25% trypsin to make them into single cells. The RPMI medium containing 0.1% fetal bovine serum was used to prepare 1x10 ^ 9 cells/L but cell suspension, and the cells were inoculated into 96 well culture plates with 100ul per well.
● Place the culture plate in the CO2 incubator and incubate it for 3-5 days at 37 ℃ and 5% CO2.
● After 3-5 days of culture, add 10 ul of MTT solution to each well, continue to incubate in the incubator for 3-6h, terminate the culture, add 10% SDS-HCl 100 ul/well, and incubate at 37 ℃ for several hours to fully dissolve MTT particles in and around cells.
● Use ELISA to measure the absorbance (OD) of each hole, and select a wavelength of 490nm.Draw cell growth curve with time as horizontal axis and OD value as vertical axis.
Use this method to measure the cell vitality. In order to ensure the accuracy of the results, it is best to measure the sticking ratio, doubling time and growth curve of each type of cell under the conditions of different number of cells inoculated before the test, and then determine the number of cells inoculated and the culture time of each hole to ensure that the culture will not be too full at the end of the culture.In addition, a blank control is set during the experiment, and the blank hole is used for zero adjustment during color comparison.
3.2.2 CCK-8 method
Cell Counting Kit-8 (CCK-8 for short) reagent can be used for simple and accurate cell proliferation and toxicity analysis.The basic principle is that the reagent contains WST-8 [chemical name: 2 - (2-methoxy-4-nitrophenyl) - 3 - (4-nitrophenyl) - 5 - (2,4-disulfonic benzene) - 2H-tetrazole monosodium salt], which is reduced by the dehydrogenase in the cell to a highly water-soluble yellow methylzan product (Formazan die) under the action of the electronic carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methody PMS).The number of generated methylzane is proportional to the number of living cells.Therefore, this property can be used directly for cell proliferation and toxicity analysis.
● Preparation of cell suspension: cell count
● Inoculate into 96 well plate: according to the appropriate number of planking cells, each hole is about 100ul cell suspension, and the same sample can be repeated 3 times.
● Culture in 37 ℃ incubator: it takes about 2-4 hours for cells to adhere to the wall after inoculation. If no adherence is required, this step can be omitted.
● Add 10ul CCK8: because the amount of CCK8 added to each hole is relatively small, there may be errors due to the reagent sticking to the hole wall. It is recommended to gently tap the culture plate after adding reagent to help mix.Or directly prepare the culture medium containing 10% CCK8, and add it in the form of liquid change.
● Culture for 1-4 hours: the amount of Formazan formed varies with different cell types.If the color development is not enough, the culture can be continued to confirm the best conditions.In particular, few blood cells form Formazan, which requires a longer color development time (5-6 hours).
● Determination of 450nm absorbance: it is recommended to use dual wavelength for determination, with detection wavelength of 450-490nm and reference wavelength of 600-650nm.
