CN113652396B - Adipose-derived mesenchymal stem cell osteogenesis inducing composition and osteogenesis inducing method - Google Patents
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Abstract
The invention discloses an osteogenic induction composition of adipose-derived mesenchymal stem cells, which consists of the following raw materials: basic culture medium, beta-disodium glycerophosphate, lithium iron phosphate, nano bismuth selenide, paulownin, fibroblast growth factor and recombinant insulin. The composition provided by the invention can effectively improve the osteogenesis proliferation differentiation efficiency of cells by adding components such as lithium iron phosphate, nano bismuth selenide, paulownin and the like. The added lithium iron phosphate and nano bismuth selenide improve the osteogenic differentiation signal activity of the adipose-derived mesenchymal stem cells, and can effectively promote the osteogenic differentiation of the cells. The paulownin and the fibroblast growth factor and the recombinant insulin together promote the proliferation of osteoblasts and shorten the time of cell differentiation. The invention also provides an osteogenic induction method of the adipose-derived mesenchymal stem cells, and the composition provided by the invention can be used for osteogenic induction differentiation of the adipose-derived mesenchymal stem cells efficiently and stably, so that the application of the adipose-derived mesenchymal stem cells in bone tissue engineering is ensured.
Description
Technical Field
The invention relates to an osteogenesis inducing composition, in particular to an osteogenesis inducing composition of adipose-derived mesenchymal stem cells and an osteogenesis inducing method.
Background
Stem cells are the original source of various tissues and organs of the body, have proliferation and differentiation capabilities, and play an important role in cell transplantation, tissue engineering and regenerative medicine along with the rapid development of medical technology. In particular, bone tissue engineering is the most rapidly developing discipline in the field of tissue engineering, and stem cells are the main seed cells of tissue engineering.
Adipose-derived mesenchymal stem cells are one of the more dominant types of stem cells. Adipose tissue is the most readily available source of mesenchymal stem cells. Adipose tissue contains a large amount of undifferentiated stem cells, and is abundant in reserves. Compared with bone marrow mesenchymal stem cells, the adipose-derived mesenchymal stem cells can be obtained from subcutaneous fat discarded in liposuction or surgery, are convenient to obtain and wide in source, and do not relate to disputes in society, ethics and the like. Adipose-derived mesenchymal stem cells can be successfully induced and differentiated into mesoderm cells such as osteoblasts, chondrocytes, skeletal muscle cells and the like under different conditions, and are ideal seed cells for tissue engineering.
However, at present, the osteogenesis of the adipose-derived mesenchymal stem cells also has some common problems of common stem cells, such as low osteogenesis induction rate, complex operation, long period, easy occurrence of adverse reactions of organisms and the like, and the problems limit the wide clinical application of the adipose-derived mesenchymal stem cells to a great extent. Therefore, it is necessary to research an osteogenesis inducer and an induction method for adipose-derived mesenchymal stem cells, which are safe and efficient, and promote the application of adipose-derived mesenchymal stem cells in bone tissue engineering.
Disclosure of Invention
In order to overcome the defects in the prior art, one of the purposes of the invention is to provide an osteogenesis inducing composition of adipose-derived mesenchymal stem cells, which effectively improves the inducing efficiency and shortens the differentiating time.
The second purpose of the invention is to provide an osteoinduction method of adipose-derived mesenchymal stem cells.
One of the purposes of the invention is realized by adopting the following technical scheme:
an osteogenic induction composition of adipose-derived mesenchymal stem cells, which consists of the following raw materials: basic culture medium, beta-disodium glycerophosphate, lithium iron phosphate, nano bismuth selenide, paulownin, fibroblast growth factor and recombinant insulin.
Further, the basal medium is DMEM/F12 medium.
Further, the concentrations of the components in the medium, in terms of the final concentration, are: beta-disodium glycerophosphate 1-5ng/mL, lithium iron phosphate 0.5-1.8ng/mL, nano bismuth selenide 2.5-4.5 ng/mL, paulownin 3-5 ng/mL, fibroblast growth factor 5-10 mug/mL, recombinant insulin 1-5 mug/mL.
Further, the concentrations of the components in the medium, in terms of the final concentration, are: beta-disodium glycerophosphate 3ng/mL, lithium iron phosphate 1.2ng/mL, nano bismuth selenide 3ng/mL, paulownin 4 ng/mL, fibroblast growth factor 8 mug/mL, recombinant insulin 3 mug/mL.
Further, the average particle size of the nano bismuth selenide is 100-200nm.
The second purpose of the invention is realized by adopting the following technical scheme:
an osteogenesis induction method of adipose-derived mesenchymal stem cells, which comprises the steps of inducing and culturing adipose-derived mesenchymal stem cells by adopting the composition to obtain osteoblasts.
Further, the adipose tissue-derived stem cells are P3-generation cells.
Further, the concentration of the adipose-derived mesenchymal stem cells in the composition is 1-5×10 4 And each mL.
Further, the induction culture conditions were: at 37 ℃,5% CO 2 Is carried out for 10-12d.
Compared with the prior art, the invention has the beneficial effects that: the invention provides an osteogenesis-inducing composition of adipose-derived mesenchymal stem cells, which is added with lithium iron phosphate, nano bismuth selenide, paulownin and other components, so that the osteogenesis proliferation differentiation efficiency of cells can be effectively improved. The added lithium iron phosphate and nano bismuth selenide improve the osteogenic differentiation signal activity of the adipose-derived mesenchymal stem cells, can effectively promote the osteogenic differentiation of the adipose-derived mesenchymal stem cells and improve the osteogenic differentiation efficiency. The paulownin and the fibroblast growth factor and the recombinant insulin together promote the proliferation of osteoblasts and shorten the time of cell differentiation.
The invention also provides an osteogenic induction method of the adipose-derived mesenchymal stem cells, and the composition provided by the invention can be used for osteogenic induction differentiation of the adipose-derived mesenchymal stem cells efficiently and stably, so that the application of the adipose-derived mesenchymal stem cells in bone tissue engineering is ensured.
Detailed Description
The present invention will be further described with reference to the following specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below.
The P3 generation adipose mesenchymal stem cells used in examples 1 to 3 were prepared by the following method:
(1) Washing the isolated adipose tissue with PBS, and adding 1% type II collagenase for shaking digestion;
(2) Centrifuging the digested solution obtained in the step (1) at 1000rpm for 15min, and collecting cell sediment at the bottom layer;
(3) Go to step2) Adding culture medium (DMEM/F12+10% FBS) to resuspension, culturing in petri dish until cell fusion degree reaches 80-90%, removing supernatant, washing with PBS, adding 0.25% pancreatin for digestion, adding DMEM/F12 culture medium to stop digestion after cell shedding, centrifuging, adding culture medium (DMEM/F12+10% FBS) again to resuspension cells, and cell density of 1×10 4 And inoculating the cells per mL into a T75 culture flask for subculturing at a ratio of 1:3, and taking P3 generation cells for experiment.
Example 1
An osteogenic induction composition of adipose-derived mesenchymal stem cells, which consists of the following raw materials: DMEM/F12 medium, beta-disodium glycerophosphate, lithium iron phosphate, nano bismuth selenide, paulownin, fibroblast growth factor, recombinant insulin; the concentration of each component in the culture medium is as follows: beta-disodium glycerophosphate 3ng/mL, lithium iron phosphate 1.2ng/mL, nano bismuth selenide 3ng/mL with an average particle size of 100nm, paulownin 4 ng/mL, fibroblast growth factor 8 mug/mL and recombinant insulin 3 mug/mL.
An osteoinduction method of adipose-derived mesenchymal stem cells, comprising the following steps: adding cultured P3 generation adipose mesenchymal stem cells into osteogenic induction composition with cell density of 1×10 4 Differentiation was induced in 6-well plates at 37℃and 5% CO per mL 2 Is carried out in the incubator of (2), the liquid is changed every two days, and the induction is continued for 10 days.
Example 2
An osteogenic induction composition of adipose-derived mesenchymal stem cells, which consists of the following raw materials: DMEM/F12 medium, beta-disodium glycerophosphate, lithium iron phosphate, nano bismuth selenide, paulownin, fibroblast growth factor, recombinant insulin; the concentration of each component in the culture medium is as follows: beta-disodium glycerophosphate 1ng/mL, lithium iron phosphate 0.5ng/mL, nano bismuth selenide 2.5 ng/mL with an average particle size of 150nm, paulownin 3ng/mL, fibroblast growth factor 5 mug/mL and recombinant insulin 1 mug/mL.
An osteoinduction method of adipose-derived mesenchymal stem cells, comprising the following steps: taking the cultured P3 generation adipose mesenchymal stemCells were added to osteoinductive compositions at a cell density of 3X 10 4 Differentiation was induced in 6-well plates at 37℃and 5% CO per mL 2 Is carried out in the incubator of (2), the liquid is changed every two days, and the induction is continued for 12 days.
Example 3
An osteogenic induction composition of adipose-derived mesenchymal stem cells, which consists of the following raw materials: DMEM/F12 medium, beta-disodium glycerophosphate, lithium iron phosphate, nano bismuth selenide, paulownin, fibroblast growth factor, recombinant insulin; the concentration of each component in the culture medium is as follows: 5ng/mL of beta-disodium glycerophosphate, 1.8ng/mL of lithium iron phosphate, 4.5 ng/mL of nano bismuth selenide with an average particle size of 200nm, 5ng/mL of paulownin, 10 mug/mL of fibroblast growth factor and 5 mug/mL of recombinant insulin.
An osteoinduction method of adipose-derived mesenchymal stem cells, comprising the following steps: adding cultured P3 generation adipose mesenchymal stem cells into osteogenic induction composition with cell density of 5×10 4 Differentiation was induced in 6-well plates at 37℃and 5% CO per mL 2 Is carried out in the incubator of (2), the liquid is changed every two days, and the induction is continued for 11 days.
Comparative example 1
Comparative example 1 provides an adipose-derived mesenchymal stem cell osteoinductive composition, differing from example 1 in that: the lithium iron phosphate was omitted and the remainder was the same as in example 1.
Comparative example 2
Comparative example 2 provides an adipose mesenchymal stem cell osteoinductive composition, differing from example 1 in that: the remainder of the procedure was the same as in example 1 except that lithium iron phosphate was replaced with lithium phosphate.
Comparative example 3
Comparative example 3 provides an adipose-derived mesenchymal stem cell osteoinductive composition, differing from example 1 in that: the nano bismuth selenide was omitted and the remainder was the same as in example 1.
Comparative example 4
Comparative example 4 provides an adipose mesenchymal stem cell osteoinductive composition, differing from example 1 in that: the nano bismuth selenide was replaced with bismuth ferrite, and the rest was the same as in example 1.
Comparative example 5
Comparative example 5 provides an adipose mesenchymal stem cell osteoinductive composition, differing from example 1 in that: the nano bismuth selenide was replaced with bismuth oxide, and the rest was the same as in example 1.
Comparative example 6
Comparative example 6 provides an adipose mesenchymal stem cell osteoinductive composition, differing from example 1 in that: the nano bismuth selenide was replaced with nano selenium, and the rest was the same as in example 1.
Comparative example 7
Comparative example 7 provides an adipose mesenchymal stem cell osteoinductive composition, differing from example 1 in that: the lithium iron phosphate was omitted and the amount of nano bismuth selenide was adjusted to 4.2 ng/mL, the remainder being the same as in example 1.
Comparative example 8
Comparative example 8 provides an adipose mesenchymal stem cell osteoinductive composition, differing from example 1 in that: the paulownin was omitted and the remainder was the same as in example 1.
The cells induced to differentiate for 10d in example 1, comparative examples 1 to 8 were discarded in the medium, washed with PBS, fixed with 40g/L paraformaldehyde at normal temperature for 10min, discarded, washed again with PBS, stained with 1g/L alizarin red at room temperature for 25min, discarded in the staining solution, washed with PBS, and observed under an inverted microscope to count the percentage of calcified area, and the results are shown in Table 1.
TABLE 1
| Group of | Calcification area (%) |
| Example 1 | 69.72 |
| Comparative example 1 | 43.51 |
| Comparative example 2 | 47.29 |
| Comparative example 3 | 40.05 |
| Comparative example 4 | 49.27 |
| Comparative example 5 | 46.62 |
| Comparative example 6 | 51.14 |
| Comparative example 7 | 48.39 |
| Comparative example 8 | 53.96 |
Cells of comparative examples 1 to 8 were induced to differentiate and cultured for 2d, 5d, 8d, and 10d, respectively, and the cells were lysed in Tris-HCl buffer pH7.4, centrifuged at 12000r/min for 5min, and the supernatant was collected, and ALP activity of each group of cells was measured using an ALP kit, and the results are shown in Table 2.
TABLE 2
| Group of | 2d | 5d | 8d | 10d |
| Example 1 | 0.37 | 0.54 | 0.67 | 0.79 |
| Comparative example 1 | 0.19 | 0.31 | 0.43 | 0.50 |
| Comparative example 2 | 0.21 | 0.32 | 0.45 | 0.54 |
| Comparative example 3 | 0.15 | 0.26 | 0.38 | 0.47 |
| Comparative example 4 | 0.23 | 0.35 | 0.49 | 0.61 |
| Comparative example 5 | 0.20 | 0.31 | 0.43 | 0.52 |
| Comparative example 6 | 0.25 | 0.37 | 0.51 | 0.64 |
| Comparative example 7 | 0.22 | 0.34 | 0.47 | 0.58 |
| Comparative example 8 | 0.28 | 0.41 | 0.55 | 0.67 |
As can be seen from tables 1 and 2, in example 1, the calcified area was large, and ALP activity was high, indicating that the osteoblasts obtained by induction differentiation in example 1 were large.
The lithium iron phosphate in the composition is omitted in comparative example 1, the lithium iron phosphate is replaced by lithium phosphate in comparative example 2, the calcified area and ALP activity of comparative example 1 are reduced more remarkably, and the fact that the addition of lithium iron phosphate in the composition can effectively promote the osteogenic differentiation of adipose-derived mesenchymal stem cells, and more osteoblasts are obtained after the composition added with the components is cultured. After replacing lithium iron phosphate with lithium phosphate, the calcified area and ALP activity were slightly improved compared to comparative example 1, but there was a gap compared to example 1, indicating that the induction effect of lithium phosphate was inferior to that of lithium iron phosphate.
The nano bismuth selenide was omitted in comparative example 3, the nano bismuth selenide was replaced with bismuth ferrite in comparative example 4, the nano bismuth selenide was replaced with bismuth oxide in comparative example 5, and the nano bismuth selenide was replaced with nano selenium in comparative example 6, and as can be seen from tables 1 and 2, the effect of inducing differentiation was inferior to that of example 1 in comparative examples 3 to 6. The nano bismuth selenide added by the invention has good effect of promoting the osteogenic differentiation of adipose-derived mesenchymal stem cells, and remarkably improves the calcified area and the ALP activity. In comparative example 7, even if the amount of nano bismuth selenide is increased after lithium iron phosphate is omitted, the osteogenic differentiation effect of the cells is inferior to that of example 1, and it is demonstrated that the synergistic effect of the two components improves the osteogenic differentiation efficiency of the adipose-derived mesenchymal stem cells.
In comparative example 8, paulownin was omitted and the calcified area and ALP activity were reduced to different extents. This is because the addition of paulownin can promote the proliferation of osteoblasts, increase the number of osteoblasts and shorten the time for cell differentiation.
In conclusion, the composition of the invention promotes the osteogenic differentiation of the adipose-derived mesenchymal stem cells by adding the components such as lithium iron phosphate, nano bismuth selenide, paulownin and the like, and effectively improves the osteogenic proliferation differentiation efficiency of the cells.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (5)
1. An osteogenic induction composition of adipose-derived mesenchymal stem cells, which is characterized by comprising the following raw materials: basic culture medium, beta-disodium glycerophosphate, lithium iron phosphate, nano bismuth selenide, paulownin, fibroblast growth factor and recombinant insulin; the basic culture medium is a DMEM/F12 culture medium; the concentration of each component in DMEM/F12 medium is as follows: beta-disodium glycerophosphate 3ng/mL, lithium iron phosphate 1.2ng/mL, nano bismuth selenide 3ng/mL with an average particle size of 100nm, paulownin 4 ng/mL, fibroblast growth factor 8 mug/mL and recombinant insulin 3 mug/mL.
2. An osteogenic induction method of adipose-derived mesenchymal stem cells, characterized in that the composition of claim 1 is used for inducing and culturing adipose-derived mesenchymal stem cells to obtain osteoblasts.
3. The method of osteoinduction of adipose mesenchymal stem cells according to claim 2, wherein the adipose mesenchymal stem cells are P3 generation cells.
4. The method for osteoinduction of adipose tissue stem cells according to claim 2, wherein the concentration of adipose tissue stem cells in the composition is 1-5 x 10 4 And each mL.
5. The method for osteoinduction of adipose-derived mesenchymal stem cells according to claim 2, wherein the induction culture conditions are: at 37 ℃,5% CO 2 Is carried out for 10-12d.
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